Hyaluronate synthase gene and uses thereof

ABSTRACT

Disclosed are DNA sequences encoding hyaluronic acid synthase that are employed to construct recombinant cells useful in the production of hyaluronate synthase and hyaluronic acid (HA). In preferred aspects, chromosomal DNA encoding the HA synthase gene, hasA, was cloned from a  Streptococcus pyogenes  genomic library. These vectors were used to transform host cells such as  E. coli  and acapsular Streptococci to produce hyaluronic acid. Resultant transformants were screened to identify colonies which have incorporated HA synthase DNA in a form that is being actively transcribed into the corresponding HA synthase enzyme. These colonies were selected and employed in the production of hyaluronic acid.

[0001] The government owns certain rights in the present inventionpursuant to grant number GM35978 from the National Institutes of Health.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to a nucleic acid encoding theenzyme hyaluronate synthase, and to the use of this nucleic acid in thepreparation of recombinant cells for the production of the hyaluronatesynthase enzyme and hyaluronic acid. Hyaluronate is also known ashyaluronic acid or hyaluronan.

[0004] 2. Description of the Related Art

[0005] The incidence of streptococcal infections is a major health andeconomic problem worldwide, particularly in developing countries (Rotta,1988). One reason for this is due to the ability of Streptococcalbacteria to grow undetected by the body's phagocytic cells (i.e.,macrophages and polymorphonuclear cells (PMNs). These cells areresponsible for recognizing and engulfing foreign microorganisms. Oneeffective way the bacteria evade surveillance is by coating themselveswith polysaccharide capsules, such as hyaluronic acid (HA) capsules.(Kendall et al., 1937). Since HA is generally nonimmunogenic (Quinn andSingh, 1957), the encapsulated bacteria do not elicit an immune responseand are, therefore, not targeted for destruction. Moreover, the capsuleexerts an antiphagocytic effect on PMNs in vitro (Hirsch, et al., 1960)and prevents attachment of Streptococcus to macrophages (Whitnack, etal., 1981). Precisely because of this, in group A and group CStreptococci, the HA capsules are major virulence factors in natural andexperimental infections (Kass, et al., 1944; Wessels, et al., 1991).Group A Streptococcus are responsible for numerous human diseasesincluding pharyngitis, impetigo, deep tissue infections, rheumatic feverand a toxic shock-like syndrome (Schaechter, et al., 1989). The group CStreptococcus equisimilis is responsible for osteomyelitis (Barson,1986), pharyngitis (Benjamin, et al., 1976), brain abscesses (Dinn,1971), and pneumonia (Rizkallah, et al., 1988; Siefkin, et al., 1983).

[0006] Structurally, HA is a high molecular weight linear polysaccharideof repeating disaccharide units consisting of N-acetylglucosamine(GlcNAc) and glucuronic acid (GlcA) (Laurent and Fraser, 1992). HA isthe only glycosaminoglycan synthesized by both mammalian and bacterialcells particularly groups A and C Streptococci. Some Streptococcusstrains make HA which is secreted into the medium as well as HAcapsules. The mechanism by which these bacteria synthesize HA is ofinterest since the production of the HA capsule is a very efficient andclever way that Streptococci use to evade surveillance by the immunesystem.

[0007] HA is synthesized by both mammalian and Streptococcus cells bythe enzyme hyaluronate synthase, that has been localized to the plasmamembrane of Streptococcus (Markovitz, et al., 1962). The synthesis of HAin these organisms is a multi-step process. Initiation involves bindingof an initial precursor, UDP-GlcNAc or UDP-GlcA. This is followed byelongation which involves alternate addition of the two sugars to thegrowing oligosaccharide chain. The growing polymer is extruded acrossthe bacterial plasma membrane region of the cell wall and into theextracellular space. Although the HA biosynthetic system was one of thefirst membrane heteropolysaccharide synthetic pathways studied, themechanism of HA synthesis is still not understood. This may be becausein vitro systems developed to date are inadequate in that de novobiosynthesis of HA has not been accomplished. Chain elongation but notnew chain initiation occurs.

[0008] The direction of HA polymer growth is a matter of disagreement.Addition of the monosaccharides could be to the reducing (Prehm, 1983)or nonreducing (Stoolmiller, et al., 1969) end of the growing HA chain.In addition, other questions that need to be addressed are (i) whethernascent chains are linked covalently to a protein, to UDP or to a lipidintermediate, (ii) whether chains are initiated using a primer, and(iii) the mechanism by which the mature polymer is extruded through theplasma membrane of the Streptococcus. Understanding the mechanism of HAbiosynthesis may allow development of alternative is strategies tocontrol Streptococcal infections by interfering in the process.

[0009] Group C S. equisimilis strain D181 synthesizes and secretes HA.Investigators have used this strain and group A strains, such as A111,to study the biosynthesis of HA and to characterize the HA-synthesizingactivity in terms of its divalent cation requirement (Stoolmiller, etal., 1969), precursor (UDP-GlcNAc and UDP-GlcUA) utilization (Ishimoto,et al., 1967; Markovitz, et al., 1959), and optimum pH (Stoolmiller, etal., 1969). The HA synthase enzyme has been studied for approximately 30years, but has not yet been identified or purified. Although a 52-kDprotein has been putatively suggested as the HA synthase (Prehm, et al.,1986), this report is now known to be in error. Furthermore, no one hassuccessfully purified to homogeneity an active enzyme. Moreover, it'snot clear whether a bona fide HA synthase molecule is all that is neededfor the generation of hyaluronic acid, or whether it might act inconcert with other cellular components or subunits. Thus, totally exvivo methods of producing HA have not been forthcoming.

[0010] Typically, HA has been prepared commercially by isolation fromeither rooster combs or extracellular media from Streptococcal cultures.One method which has been developed for preparing HA is through the useof cultures of HA-producing streptococcal bacteria. U.S. Pat. No.4,517,295, describes such a procedure, wherein HA-producing Streptococciare fermented under anaerobic conditions in a CO₂-enriched growthmedium. Under these conditions, HA is produced and can be extracted fromthe broth. It is generally felt that isolation of HA from rooster combis laborious and difficult, since one starts with HA in a less purestate. The advantage of isolation from rooster comb is that the HAproduced is of higher molecular weight. However, preparation of HA bybacterial fermentation is easier, since the HA is of higher purity tostart with. Usually, however, the molecular weight of HA produced inthis way is smaller than that from rooster combs. Therefore, a techniquethat would allow the production of high molecular weight HA by bacterialfermentation would be an improvement over existing procedures.

[0011] High molecular weight HA has a wide variety of usefulapplications—ranging from cosmetics to eye surgery (Laurent and Fraser,1992). Due to its potential for high viscosity and its highbiocompatibility, HA finds particular application in eye surgery as areplacement for vitreous fluid. HA has also been used to treatracehorses for traumatic arthritis by intra-articular injections of HA,in shaving cream as a lubricant, and in a variety of cosmetic productsdue to its physiochemical properties of high viscosity and its abilityto retain moisture for long periods of time. In general, the highermolecular weight the HA that is employed the better. This is because HAsolution viscosity increases with the average molecular weight of theindividual HA polymer molecules in the solution. Unfortunately, veryhigh molecular weight HA, such as that ranging up to 10⁷, has beendifficult to obtain by currently available isolation procedures.

[0012] To address these or other difficulties, there is a need for newmethods and constructs that can be used to produce HA having one or moreimproved properties such as greater purity or ease of preparation. Inparticular, there is a need to develop methodology for the production oflarger amounts of relatively higher molecular weight and purity HA thanis available from current technology. The present invention addressesone or more shortcomings in the art through the application ofrecombinant DNA technology.

SUMMARY OF THE INVENTION

[0013] The present invention involves the application of recombinant DNAtechnology to solving one or more problems in the art of hyaluronic acidpreparation. These problems are addressed through the isolation and useof a DNA segment encoding all or a portion of the hyaluronate synthasegene, the gene responsible for HA chain biosynthesis. The gene wascloned from DNA of an appropriate microbial source and engineered intouseful recombinant constructs for the preparation of HA and for thepreparation of large quantities of the HA synthase enzyme itself.

[0014] The present invention, in a general and overall sense, concernsthe isolation and characterization of a hyaluronate or hyaluronic acidsynthase gene, as may be used for the polymerization of glucuronic acidand N-acetylglucosamine into the glycosaminoglycan hyaluronic acid. Thepresent inventors have identified the hasA locus and have determined thesequence encoding the Hyaluronic acid synthase (HA synthase) gene fromStreptococcus. The hasA gene product, HasA, has been expressed inhomologous and heterologous cells, can be used to isolate hyaluronicacid synthase, and can be used for the production of hyaluronic acid.The hasA gene also provides a new probe to assess the potential ofbacterial specimens to produce hyaluronic acid.

[0015] The present invention encompasses a novel gene, hasA. Theexpression of this gene correlates with virulence of Streptococcalstrains, by providing a means of escaping immune surveillance. The term,“hyaluronic acid synthase”, “hyaluronate synthase”, “hyaluronansynthase” and “HA synthase”, are used interchangeably to describe anenzyme that polymerizes a glycosaminoglycan polysaccharide chaincomposed of alternating glucuronic acid and N-acetylglucosamine sugars.

[0016] Through the application of techniques and knowledge set forthherein, those of skill in the art will be able to obtain nucleic acidsegments encoding an HA synthase gene. Through isolation of the HA gene,from whatever source is chosen, one will have the ability to realizesignificant advantages such as an ability to manipulate the host that ischosen to express the HA synthase gene, the fermentation environmentchosen for HA production, as well as genetic manipulation of theunderlying gene. As those of skill in the art will recognize, in lightof the present disclosure, this will provide additional significantadvantages both in the ability to control the expression of the gene andin the nature of the gene product that is realized.

[0017] Accordingly, the invention is directed to the isolation of DNAthat comprises the HA synthase gene, whether it be from prokaryotic oreukaryotic sources. This is possible because the enzyme, and indeed thegene, is one found in both eukaryotes and some prokaryotes. Typicalprokaryotic sources will include Group A or Group C Streptococcussources such as S. pyogenes, S. equisimilis, or S. zooepidemicus.Eukaryotes are also known to produce HA (Ng and Schwartz, 1989) and thushave HA synthase genes that may be employed in connection with theinvention. For example, it is known that HA is produced in rooster combsby mesodermal cells of the rooster. These cells can be employed toisolate starting mRNA for the production of cDNA libraries by well knowntechniques, which can subsequently be screened by novel screeningtechniques set forth herein. Other eukaryotic sources that can beemployed include synovial chondrocytes and fibroblasts, dermalfibroblasts, and even trabecular-meshwork cells of the eye.

[0018] HA synthase-encoding nucleic acid segments of the presentinvention are defined as being isolated free of total chromosomal orgenomic DNA such that they may be readily manipulated by recombinant DNAtechniques. Accordingly, as used herein, the phrase “substantiallypurified DNA segment” refers to a DNA segment isolated free of totalchromosomal or genomic DNA and retained in a state rendering it usefulfor the practice of recombinant techniques, such as DNA in the form of adiscrete isolated DNA fragment, or a vector (e.g., plasmid, phage orvirus) incorporating such a fragment.

[0019] A preferred embodiment of the present invention is a purifiednucleic acid segment encoding HA synthase, wherein the segment encodes aprotein having an amino acid sequence in accordance with SEQ ID NO:2, orthat is capable of hybridizing to the nucleotide sequence of SEQ ID NO:1under standard hybridization conditions as described herein. Thenucleotide segment of the present invention is a purified nucleic acidsegment, further defined as including a nucleotide sequence as shown inFIG. 7, and in accordance with SEQ ID NO:1.

[0020] In a more preferred embodiment the purified nucleic acid segmentconsists essentially of the nucleotide sequence of SEQ ID NO:1. As usedherein, the term “nucleic acid segment” and “DNA segment” are usedinterchangeably and refer to a DNA molecule which has been isolated freeof total genomic DNA of a particular species. Therefore, a “purified”DNA or nucleic acid segment as used herein, refers to a DNA segmentwhich contains an hasA coding sequence yet is isolated away from, orpurified free from, total genomic DNA, for example, total Streptococcuspyogenes or, for example, mammalian host genomic DNA. Included withinthe term “DNA segment”, are DNA segments and smaller fragments of suchsegments, and also recombinant vectors, including, for example,plasmids, cosmids, phage, viruses, and the like.

[0021] Similarly, a DNA segment comprising an isolated or purified hasAgene refers to a DNA segment including hasA coding sequences isolatedsubstantially away from other naturally occurring genes or proteinencoding sequences. In this respect, the term “gene” is used forsimplicity to refer to a functional protein, polypeptide or peptideencoding unit. As will be understood by those in the art, thisfunctional term includes genomic sequences, cDNA sequences orcombinations thereof. “Isolated substantially away from other codingsequences” means that the gene of interest, in this case hasA, forms thesignificant part of the coding region of the DNA segment, and that theDNA segment does not contain large portions of naturally-occurringcoding DNA, such as large chromosomal fragments or other functionalgenes or DNA coding regions. Of course, this refers to the DNA segmentas originally isolated, and does not exclude genes or coding regionslater added to, or intentionally left in the segment by the hand of man.

[0022] Due to certain advantages associated with the use of prokaryoticsources, one will likely realize the most advantages upon isolation ofthe HA synthase gene from prokaryotes such as S. pyogenes or S.equisimilis. One such advantage is that, typically, eukaryotic enzymesmay require significant post-translational modifications that can onlybe achieved in a eukaryotic host. This will tend to limit theapplicability of any eukaryotic HA synthase gene that is obtained.Moreover, those of skill will likely realize additional advantages interms of time and ease of genetic manipulation where a prokaryoticenzyme gene is sought to be employed. These additional advantagesinclude (a) the ease of isolation of a prokaryotic gene because of therelatively small size of the genome and, therefore, the reduced amountof screening of the corresponding genomic library and (b) the ease ofmanipulation because the overall size of the coding region of aprokaryotic gene is significantly smaller due to the absence of introns.Furthermore, if the product of the HA synthase gene (i.e., the enzyme)requires posttranslational modifications, these would best be achievedin a similar prokaryotic cellular environment (host) from which the genewas derived.

[0023] Preferably, DNA sequences in accordance with the presentinvention will further include genetic control regions which allow theexpression of the sequence in a selected recombinant host. Of course,the nature of the control region employed will generally vary dependingon the particular use (e.g., cloning host) envisioned. For example, instreptococcal hosts, the preferred control region is the homologouscontrol region associated with the structural gene in its natural state.The homologous control region, in fact, may be coisolated directly withthe isolation of the HA synthase structural gene itself through thepractice of certain preferred techniques disclosed herein.

[0024] In particular embodiments, the invention concerns isolated DNAsegments and recombinant vectors incorporating DNA sequences whichencode an hasA gene, that includes within its amino acid sequence anamino acid sequence in accordance with SEQ ID NO:2. Moreover, in otherparticular embodiments, the invention concerns isolated DNA segments andrecombinant vectors incorporating DNA sequences which encode a gene thatincludes within its amino acid sequence the amino acid sequence of anhasA gene corresponding to Streptococcus pyogenes hasA. Naturally, wherethe DNA segment or vector encodes a full length HasA protein, or isintended for use in expressing the HasA protein, the most preferredsequences are those which are essentially as set forth in SEQ ID NO:2.

[0025] Nucleic acid segments having HA synthase activity may be isolatedby the methods described hereinabove. The term “a sequence essentiallyas set forth in SEQ ID NO:2” means that the sequence substantiallycorresponds to a portion of SEQ ID NO:2 and has relatively few aminoacids which are not identical to, or a biologically functionalequivalent of, the amino acids of SEQ ID NO:2. The term “biologicallyfunctional equivalent” is well understood in the art and is furtherdefined in detail herein, as a gene having a sequence essentially as setforth in SEQ ID NO:2, and that is associated with the ability ofStreptococcus to produce HA and a hyaluronic acid coat. Accordingly,sequences which have between about 70% and about 80%; or morepreferably, between about 81% and about 90%; or even more preferably,between about 91% and about 99%; of amino acids which are identical orfunctionally equivalent to the amino acids of SEQ ID NO:2 will besequences which are “essentially as set forth in SEQ ID NO:2”.

[0026] Another preferred embodiment of the present invention is apurified nucleic acid segment that encodes a protein in accordance withSEQ ID NO:2, further defined as a recombinant vector. As used herein theterm, “recombinant vector”, refers to a vector that has been modified tocontain a nucleic acid segment that encodes an HasA protein, or fragmentthereof. The recombinant vector may be further defined as an expressionvector comprising a promoter operatively linked to said HasA encodingnucleic acid segment.

[0027] A further preferred embodiment of the present invention is a hostcell, made recombinant with a recombinant vector comprising an hasAgene. The preferred recombinant host cell may be a prokaryotic cell. Inanother embodiment, the recombinant host cell is a eukaryotic cell. Asused herein, the term “engineered” or “recombinant” cell is intended torefer to a cell into which a recombinant gene, such as a gene encodinghasA, has been introduced. Therefore, engineered cells aredistinguishable from naturally occurring cells which do not contain arecombinantly introduced gene. Engineered cells are thus cells having agene or genes introduced through the hand of man. Recombinantlyintroduced genes will either be in the form of a cDNA gene, a copy of agenomic gene, or will include genes positioned adjacent to a promoternot naturally associated with the particular introduced gene.

[0028] Where one desires to use a host other than Streptococcus, as maybe used to produce recombinant HA synthase, it may be advantageous toemploy a prokaryotic system such as E. coli, B. subtilis, Lactococcussp., or even eukaryotic systems such as yeast or Chinese hamster ovary,African green monkey kidney cells, VERO cells, or the like. Of course,where this is undertaken, it will generally be desirable to bring the HAsynthase gene under the control of sequences which are functional in theselected alternative host. The appropriate DNA control sequences, aswell as their construction and use, are generally well known in the artas discussed in more detail herein below.

[0029] In preferred embodiments, the HA synthase-encoding DNA segmentsfurther include DNA sequences, known in the art functionally as originsof replication or “replicons”, which allow replication of contiguoussequences by the particular host. Such origins allow the preparation ofextrachromosomally localized and replicating chimeric segments orplasmids, to which HA synthase DNA sequences are ligated. In morepreferred instances, the employed origin is one capable of replicationin Streptococcus hosts. However, for more versatility of cloned DNAsegments, it may be desirable to alternatively or even additionallyemploy origins recognized by other host systems whose use iscontemplated (such as in a shuttle vector).

[0030] The isolation and use of other replication origins such as theSV40, polyoma or bovine papilloma virus origins, which may be employedfor cloning in a number of higher organisms, are well known (Fiers, etal., 1978). In certain embodiments, the invention may thus be defined interms of a recombinant transformation vector which includes the HAsynthase gene sequence together with an appropriate replication originand under the control of selected control regions.

[0031] In accordance with the present invention, the HA synthase gene,when from a prokaryotic source such as a Streptococcal source, isobtained by the following general steps. First, the genetic loci areidentified by transposon insertional mutagenesis. One such transposonsystem is the TN916, obtainable from the transposon donor strain E.faecalis CG110, which was used to mutate the mucoid strain ofStreptococcus pyogenes S43. Mutants were isolated and the genomic DNAsurrounding the transposon was sequenced and used to deriveoligonucleotides for use in cloning the wild-type gene. Phage librarieswere screened, and two clones, λ1X and λ2Y, were obtained that containedthe predicted sequence. The locus was characterized by restrictionmapping and southern blot analysis.

[0032] Thus, although the present invention is exemplified in terms ofclones screened via transposon mediated mutagenesis, it will beappreciated by those of skill in the art that other means may be used toobtain the hasA gene, in light of the present disclosure. For example,polymerase chain reaction produced DNA fragments may be obtained whichcontain full complements of genes from a number of sources, includingother strains of Streptococcus or from eukaryotic sources, such as cDNAlibraries. Virtually any molecular cloning approach may be employed forthe generation of DNA fragments in accordance with the presentinvention. Thus, the only limitation generally on the particular methodemployed for DNA isolation is that the isolated nucleic acids shouldencode a biologically functional equivalent HA synthase.

[0033] Once the DNA has been isolated it is ligated together with aselected vector. Virtually any cloning vector can be employed to realizeadvantages in accordance with the invention. Typical useful vectorsinclude plasmids and phages for use in prokaryotic organisms and evenviral vectors for use in eukaryotic organisms. Examples includepBluescript™, pSA3, lambda, SV40, polyoma, adenovirus, bovine papillomavirus and retroviruses. However, it is believed that particularadvantages will ultimately be realized where vectors capable ofreplication in both Lactococcus or Bacillus strains and E. coli areemployed.

[0034] Vectors such as these, exemplified by the pSA3 vector of Dao andFerretti (Dao, et al., 1985) or the pAT19 vector of Trieu-Cuot, et al.(1991), allow one to perform clonal colony selection in an easilymanipulated host such as E. coli, followed by subsequent transfer backinto a Lactococcus or Bacillus strain for production of HA. This isadvantageous in that one can augment the Lactococcus or Bacillusstrain's ability to synthesize HA through gene dosaging (i.e., providingextra copies of the HA synthase gene by amplification) and/or inclusionof additional genes to increase the availability of HA precursors. Theinherent ability of Streptococci to synthesize HA can also be augmentedthrough the formation of extra copies, or amplification, of the plasmidthat carries the HA synthase gene. This amplification can account for upto a 10-fold increase in plasmid copy number and, therefore, the HAsynthase gene copy number.

[0035] Another procedure that would further augment HA synthase genecopy number is the insertion of multiple copies of the gene into theplasmid. This extra amplification would be especially feasible, sincethe bacterial HA synthase gene size is small. In any event, thechromosomal DNA-ligated vector is employed to transfect the host that isselected for clonal screening purposes such as E. coli, through the useof a vector that is capable of expressing the inserted DNA in the chosenhost.

[0036] Where a eukaryotic source such as dermal or synovial fibroblastsor rooster comb cells is employed, one will desire to proceed initiallyby preparing a cDNA library. This is carried out first by isolation ofmRNA from the above cells, followed by preparation of double strandedcDNA using an enzyme with reverse transcriptase activity and ligationwith the selected vector. Numerous possibilities are available and knownin the art for the preparation of the double stranded cDNA, and all suchtechniques are believed to be applicable. A preferred technique involvesreverse transcription. Once a population of double stranded cDNAs isobtained, a cDNA library is prepared in the selected host by acceptedtechniques, such as by ligation into the appropriate vector andamplification in the appropriate host. Due to the high number of clonesthat are obtained, and the relative ease of screening large numbers ofclones by the techniques set forth herein, one may desire to employphage expression vectors, such as λgt11 or λgt12, for the cloning andexpression screening of cDNA clones.

[0037] Due to the general absence of correct information regarding theHA synthase enzyme, traditional approaches to clonal screening, such asolgonucleotide hybridization or immunological screening, was notavailable. Accordingly, it was necessary for the inventors to use analternative approach based on phenotype to screen and select for the HAsynthase that rely on abrogating expression of HA synthase activity. Themethods which were developed can be applies to screen the selected hostregardless of whether a eukaryotic or prokaryotic gene is sought. Onemethod involves the application of a dye exclusion technique to identifyclones which contain HA. The typical dye employed, India ink, isexcluded from an HA capsule and allows visualization of HA by negativestaining. A second method involves positive staining, such as withAlcian Blue, to identify HA producing clones. Alcian Blue binds to andstains polyanionic molecules such as HA (Scott, et al., 1964). However,in that India ink or Alcian Blue is not entirely specific for HA, thepresent inventors employed additional screening methods as described inExamples I and II.

[0038] A variety of additional screening and validation procedures arealso set forth herein that can variously be employed to identify thepresence of either the HA enzyme or its HA product as a means foridentifying positive clones or negative clones (mutants). Theseprocedures included the use of Percoll gradient centrifugation and theability of membrane fractions from candidate clones to incorporateauthentic radiolabeled sugar nucleotides into high molecular weight HA.

[0039] In certain other embodiments, the invention concerns isolated DNAsegments and recombinant vectors that include within their sequence anucleic acid sequence essentially as set forth in SEQ ID NO:1. The term“essentially as set forth in SEQ ID NO:1”, is used in the same sense asdescribed above and means that the nucleic acid sequence substantiallycorresponds to a portion of SEQ ID NO:1, and has relatively few codonswhich are not identical, or functionally equivalent, to the codons ofSEQ ID NO:1. The term “functionally equivalent codon” is used herein torefer to codons that encode the same amino acid, such as the six codonsfor arginine or serine, as set forth in Table I, and also refers tocodons that encode biologically equivalent amino acids.

[0040] It will also be understood that amino acid and nucleic acidsequences may include additional residues, such as additional N- orC-terminal amino acids or 5′ or 3′ sequences, and yet still beessentially as set forth in one of the sequences disclosed herein, solong as the sequence meets the criteria set forth above, including themaintenance of biological protein activity where protein expression andenzyme activity is concerned. The addition of terminal sequencesparticularly applies to nucleic acid sequences which may, for example,include various non-coding sequences flanking either of the 5′ or 3′portions of the coding region or may include various internal sequences,which are known to occur within genes.

[0041] Allowing for the degeneracy of the genetic code, sequences whichhave between about 70% and about 80%; or more preferably, between about80% and about 90%; or even more preferably, between about 90% and about99%; of nucleotides which are identical to the nucleotides of SEQ IDNO:1 will be sequences which are “essentially as set forth in SEQ IDNO:1”. Sequences which are essentially the same as those set forth inSEQ ID NO:1 may also be functionally defined as sequences which arecapable of hybridizing to a nucleic acid segment containing thecomplement of SEQ ID NO:1 under relatively stringent conditions.Suitable relatively stringent hybridization conditions will be wellknown to those of skill in the art and are clearly set forth herein, forexample conditions for use with southern and northern blot analysis.

[0042] The term “standard hybridization conditions” as used herein, isused to describe those conditions under which substantiallycomplementary nucleic acid segments will form standard Watson-Crickbase-pairing. A number of factor are known that determine thespecificity of binding or hybridization, such as pH, salt concentration,the presence of chaotropic agents (e.g. formamide and dimethylsulfoxide), the length of the segments that are hybridizing, and thelike.

[0043] For use with the present invention, standard hybridizationconditions for relatively large segments, that is segments longer thanabout 100 nucleotides, will include a hybridization mixture havingbetween about 0.3 to 0.6 M NaCl, a divalent cation chelator (e.g. EDTAat about 0.05 mM to about 0.5 mM), and a buffering agent (e.g. Na2PO4 atabout 10 mM to 100 mM, pH 7.2), at a temperature of about 65° C. Thepreferred conditions for hybridization are a hybridization mixturecomprising 0.5 M NaCl, 5 mM EDTA, 0.1 M Na₂PO₄, pH 7.2 and 1% N-laurylsarcosine, at a temperature of 65° C. Naturally, conditions that affectthe hybridization temperature, such as the addition of chaotropicagents, such as formamide, will be known to those of skill in the art,and are encompassed by the present invention.

[0044] When it is contemplated that shorter nucleic acid segments willbe used for hybridization, for example fragments between about 14 andabout 100 nucleotides, salt and temperature conditions will be alteredto increase the specificity of nucleic acid segment binding. Preferredconditions for the hybridization of short nucleic acid segments includelowering the hybridization temperature to about 37° C., and increasingthe salt concentration to about 0.5 to 1.5 M NaCl with 1.5 M NaCl beingparticularly preferred. TABLE I CODON DEGENERACY Amino Acids CodonsAlanine Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGU Aspartic acid Asp DGAC GAU Glutamic acid Glu E GAA GAG Phenylalanine Phe F UUC UUU GlycineGly G GGA GGC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA AUCAUU Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUUMethionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCGCCU Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGUSerine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACUValine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

[0045] Naturally, the present invention also encompasses DNA segmentswhich are complementary, or essentially complementary, to the sequenceset forth in SEQ ID NO:1. Nucleic acid sequences which are“complementary” are those which are capable of base-pairing according tothe standard Watson-Crick complementarity rules. As used herein, theterm “complementary sequences” means nucleic acid sequences which aresubstantially complementary, as may be assessed by the same nucleotidecomparison set forth above, or as defined as being capable ofhybridizing to the nucleic acid segment of SEQ ID NO:1 under relativelystringent conditions such as those described herein in Example III.

[0046] The nucleic acid segments of the present invention, regardless ofthe length of the coding sequence itself, may be combined with other DNAsequences, such as promoters, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, other coding segments,and the like, such that their overall length may vary considerably. Itis therefore contemplated that a nucleic acid fragment of almost anylength may be employed, with the total length preferably being limitedby the ease of preparation and use in the intended recombinant DNAprotocol. For example, nucleic acid fragments may be prepared whichinclude a short stretch complementary to SEQ ID NO:1, such as about 10to 15 or 20, 30, or 40 or so nucleotides, and which are up to 10,000 or5,000 base pairs in length, with segments of 3,000 being preferred incertain cases. DNA segments with total lengths of about 1,000, 500, 200,100 and about 50 base pairs in length are also contemplated to beuseful.

[0047] A preferred embodiment of the present invention is a nucleic acidsegment which comprises at least a 10-14 nucleotide long stretch whichcorresponds to, or is complementary to, the nucleic acid sequence of SEQID NO:1. In a more preferred embodiment the nucleic acid is furtherdefined as comprising at least a 20 nucleotide long stretch, a 30nucleotide long stretch, 50 nucleotide long stretch, 100 nucleotide longstretch, a 200 nucleotide long stretch, a 500 nucleotide long stretch, a1000 nucleotide long stretch, a 1500 nucleotide long stretch, or atleast a 1441 nucleotide long stretch which corresponds to, or iscomplementary to, the nucleic acid sequence of SEQ ID NO:1. The nucleicacid segment may be further defined as having the nucleic acid sequenceof SEQ ID NO:1.

[0048] A related embodiment of the present invention is a nucleic acidsegment which comprises at least a 10-14 nucleotide long stretch whichcorresponds to, or is complementary to, the nucleic acid sequence of SEQID NO:1, further defined as comprising a nucleic acid fragment of up to10,000 basepairs in length. A more preferred embodiment if a nucleicacid fragment comprising from 14 nucleotides of SEQ ID NO:1 up to 5,000basepairs in length, 3,000 basepairs in length, 1,000 basepairs inlength, 500 basepairs in length, or 100 basepairs in length.

[0049] Naturally, it will also be understood that this invention is notlimited to the particular nucleic acid and amino acid sequences of SEQID NOS:1 and 2. Recombinant vectors and isolated DNA segments maytherefore variously include the hasA coding regions themselves, codingregions bearing selected alterations or modifications in the basiccoding region, or they may encode larger polypeptides which neverthelessinclude hasA-coding regions or may encode biologically functionalequivalent proteins or peptides which have variant amino acidssequences.

[0050] The DNA segments of the present invention encompass biologicallyfunctional equivalent HasA proteins and peptides. Such sequences mayarise as a consequence of codon redundancy and functional equivalencywhich are known to occur naturally within nucleic acid sequences and theproteins thus encoded. Alternatively, functionally equivalent proteinsor peptides may be created via the application of recombinant DNAtechnology, in which changes in the protein structure may be engineered,based on considerations of the properties of the amino acids beingexchanged. Changes designed by man may be introduced through theapplication of site-directed mutagenesis techniques, e.g., to introduceimprovements to the enzyme activity or to antigenicity of the HasAprotein or to test HasA mutants in order to examine HA synthase activityat the molecular level.

[0051] A preferred embodiment of the present invention is a purifiedcomposition comprising a polypeptide having an amino acid sequence inaccordance with SEQ ID NO:2. The term “purified” as used herein, isintended to refer to an HasA protein composition, wherein the HasAprotein is purified to any degree relative to its naturally-obtainablestate, i.e., in this case, relative to its purity within a prokaryoticcell extract. A preferred cell for the isolation of HasA protein is aStreptococcus pyogenes cell, however, HasA protein may also be isolatedfrom other members of the Streptococcus genus, patient specimens,recombinant cells, infected tissues, isolated subpopulation of tissuesthat contain high levels of hyaluronate in the extracellular matrix, andthe like, as will be known to those of skill in the art, in light of thepresent disclosure. A purified HasA protein composition therefore alsorefers to a polypeptide having the amino acid sequence of SEQ ID NO:2,free from the environment in which it may naturally occur.

[0052] If desired, one may also prepare fusion proteins and peptides,e.g., where the HasA protein coding regions are aligned within the sameexpression unit with other proteins or peptides having desiredfunctions, such as for purification or immunodetection purposes (e.g.,proteins which may be purified by affinity chromatography and enzymelabel coding regions, respectively).

[0053] Turning to the expression of the hasA gene whether from genomicDNA, or a cDNA one may proceed to prepare an expression system for therecombinant preparation of HasA protein. The engineering of DNAsegment(s) for expression in a prokaryotic or eukaryotic system may beperformed by techniques generally known to those of skill in recombinantexpression. For example, one may prepare a HasA-GST(glutathione-S-transferase) fusion protein that is a convenient means ofbacterial expression. However, it is believed that virtually anyexpression system may be employed in the expression of HasA.

[0054] HasA may be successfully expressed in eukaryotic expressionsystems, however, the inventors aver that bacterial expression systemscan be used for the preparation of HasA for all purposes. The cDNA forHasA may be separately expressed in bacterial systems, with the encodedproteins being expressed as fusions with β-galactosidase, avidin,ubiquitin, Schistosoma japonicum glutathione S-transferase,maltose-binding protein, polyhistidine-tags, epitope-tags (e.g., myc andFLAG) and the like. It is believed that bacterial expression willultimately have advantages over eukaryotic expression in terms of easeof use and quantity of materials obtained thereby.

[0055] It is proposed that transformation of host cells with DNAsegments encoding HasA will provide a convenient means for obtaining anHasA protein. It is also proposed that cDNA, genomic sequences, andcombinations thereof, are suitable for eukaryotic expression, as thehost cell will, of course, process the genomic transcripts to yieldfunctional mRNA for translation into protein.

[0056] Another embodiment of the present invention is a method ofpreparing a protein composition comprising growing recombinant host cellcomprising a vector that encodes a protein which includes an amino acidsequence in accordance with SEQ ID NO:2. The host cell will be grownunder conditions permitting nucleic acid expression and proteinproduction followed by recovery of the protein so produced. Theproduction of HA synthase and HA, including: the host cell, conditionspermitting nucleic acid expression, protein production and recovery willbe known to those of skill in the art in light of the present disclosureof the hasA gene, and the hasA gene protein product HasA, and by themethods described in Examples III, IV, and V.

[0057] Preferred hosts for the expression of hyaluronic acid areprokaryotes, such as S. pyogenes, S. eguisimilis, and other suitablemembers of the Streptococcus species. However, it is also contemplatedthat HA may be synthesized by heterologous host cells expressing HAsynthase, such as species members of the Bacillus, Salmonella,Pseudomonas, Enterococcus, or even Escherichia genus. A most preferredhost for expression of the HA synthase of the present invention is abacteria transformed with the hasA gene of the present invention, suchas Lactococcus, Bacillus subtilis or S. pyogenes.

[0058] It is similarly believed that almost any eukaryotic expressionsystem may be utilized for the expression of hasA e.g.,baculovirus-based, glutamine synthase-based, dihydrofolatereductase-based systems, SV-40 based, adenovirus-based,cytomegalovirus-based, and the like, could be employed. For expressionin this manner, one would position the coding sequences adjacent to andunder the control of the promoter. It is understood in the art that tobring a coding sequence under the control of such a promoter, onepositions the 5′ end of the transcription initiation site of thetranscriptional reading frame of the protein between about 1 and about50 nucleotides “downstream” of (i.e., 3′ of) the chosen promoter.

[0059] Where eukaryotic expression is contemplated, one will alsotypically desire to incorporate into the transcriptional unit whichincludes the hasA gene, an appropriate polyadenylation site (e.g.,5′-AATAAA-3′) if one was not contained within the original clonedsegment. Typically, the poly A addition site is placed about 30 to 2000nucleotides “downstream” of the termination site of the protein at aposition prior to transcription termination.

[0060] It is contemplated that virtually any of the commonly employedhost cells can be used in connection with the expression of hasA inaccordance herewith. Examples of preferred cell lines for expressing theHA synthase gene of the present invention include cell lines typicallyemployed for eukaryotic expression such as 239, AtT-20, HepG2, VERO,HeLa, CHO, WI 38, BHK, COS-7, RIN and MDCK cell lines.

[0061] This will generally include the steps of providing a recombinanthost bearing the recombinant DNA segment encoding the HA synthase enzymeand capable of expressing the enzyme; culturing the recombinant host inmedia under conditions that will allow for transcription of the clonedHA gene and appropriate for the production of the hyaluronic acid; andis separating and purifying the HA synthase enzyme or the secretedhyaluronic acid from the recombinant host.

[0062] Generally, the conditions appropriate for expression of thecloned HA synthase gene will depend upon the promoter, the vector, andthe host system that is employed. For example, where one employs the lacpromoter, one will desire to induce transcription through the inclusionof a material that will stimulate lac transcription, such as IPTG. Whereother promoters are employed, different materials may be needed toinduce or otherwise up-regulate transcription. In addition, to obtainingexpression of the synthase, one will preferably desire to provide anenvironment that is conducive to HA synthesis by including appropriategenes encoding enzymes needed for the biosynthesis of sugar nucleotideprecursors, and by using growth media containing substrates for theprecursor-supplying enzymes, such N-acetylglucosamine (GlcNAc) andglucose (Glc).

[0063] One may further desire to incorporate the gene in a host which isdefective in the enzyme hyaluronidase, so that the product synthesizedby the enzyme will not be degraded in the medium. Furthermore, a hostwould be chosen to optimize production of HA. For example, a suitablehost would be one that produced large quantities of the sugar nucleotideprecursors to support the HA synthase enzyme and allow it to producelarge quantities of HA. Such a host may be found naturally or may bemade by a variety of techniques including mutagenesis or recombinant DNAtechnology. The genes for the sugar nucleotide synthesizing enzymes,particularly the UDP-Glc dehydrogenase required to produce UDP-GlcA,could also be isolated and incorporated in a vector along with the HAsynthase gene. A preferred embodiment of the present invention is a hostcontaining these ancillary recombinant genes and the amplification ofthese gene products thereby allowing for increased production of HA.

[0064] In the case where production of HA synthase is desired, theenzyme is preferably synthesized in bacteria using the T7 expressionsystem (Studier, et al., 1990). pT5 plasmids containing the HA synthasegene inserted adjacent to the phi10 promoter are transformed into E.coli stain BL21(DE3)pLysS. In this strain the T7 gene encoding thebacteriophage RNA polymerase is under control of the E. coli lacZpromoter. Therefore, the polymerase can be induced by IPTG andtranscription of the HA synthase gene is, in turn, induced from the φ10promoter of the pT5 vector.

[0065] The means employed for culturing of the host cell is not believedto be particularly crucial. For useful details, one may wish to refer tothe disclosure of U.S. Pat. Nos. 4,517,295; 4,801,539; 4,784,990; or4,780,414; all incorporated herein by reference. Where a prokaryotichost is employed, such as S. pyogenes or S. equisimilis, one may desireto employ a fermentation of the bacteria under anaerobic conditions inCO₂-enriched broth growth media. This allows for a greater production ofHA than under aerobic conditions. Another consideration is thatStreptococcal cells grown anaerobically do not produce pyrogenicexotoxins. Appropriate growth conditions can be customized for otherprokaryotic hosts, as will be known to those of skill in the art, inlight of the present disclosure.

[0066] Once the appropriate host has been constructed, and culturedunder conditions appropriate for the production of HA, one will desireto separate the HA so produced. Typically, the HA will be secreted orotherwise shed by the recombinant organism into the surrounding media,allowing the ready isolation of HA from the media by known techniques.For example, HA can be separated from the media by filtering and/or incombination with precipitation by alcohols such as ethanol. Otherprecipitation agents include organic solvents such as acetone orquaternary organic ammonium salts such as cetyl pyridinium chloride(CPC).

[0067] A preferred technique for isolation of HA is described in U.S.Pat. No. 4,517,295 in which the organic carboxylic acid, trichloroaceticacid, is added to the bacterial suspension at the end of thefermentation. The trichloroacetic acid causes the bacterial cells toclump and die and facilitates the ease of separating these cells andassociated debris from HA, the desired product. The clarifiedsupernatant is concentrated and dialyzed to remove low molecular weightcontaminants including the organic acid. The aforementioned procedureutilizes Millipore(tm) filtration through filter cassettes containing0.22 μm pore size filters. Diafiltration is continued until theconductivity of the solution decreases to approximately 0.5 mega-ohms.

[0068] The concentrated HA is precipitated by adding an excess ofreagent grade ethanol or other organic solvent and the precipitated HAis then dried by washing with ethanol and vacuum dried, lyophilized orspray dried to remove alcohol. The HA can then be redissolved in aborate buffer, pH 8, and precipitated with CPC or certain other organicammonium salts such as CETAB, a mixed trimethyl ammonium bromidesolution at 4 degree(s) C. The precipitated HA is recovered by coarsefiltration, resuspended in 1 M NaCl, diafiltered and concentrated asfurther described in the above referenced patent. The resultant HA isfilter sterilized and ready to be converted to an appropriate salt, drypowder or sterile solution, depending on the desired end use.

BRIEF DESCRIPTION OF THE DRAWINGS

[0069] The following drawings form part of the present specification andare included to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

[0070]FIG. 1: Restriction map of the streptococcal HA biosynthesislocus. EcoRI (E) restriction sites on the wild-type S43 chromosome areillustrated with respect to the relevant Tn insertion and the minimalextent of the associated deletion (hatched box). The region of DNAcapable of directing HA synthesis in mutants and heterologous species,pPD41Δ5 is shown. Two HindIII (H), two PstI (P) and one BglII (B) siteswere found. KpnI, BamHI, SalI, SacI, SmaI, SphI, and XbaI did not cutthe pPD41Δ5 insert. The multiple cloning site (M) is the result offusion of the deleted, blunt ended DNA and the pAT19 M. The inserts ofthe initial clones pB3 and pPD41 are shown above the genomic map. Thelarge EcoRI fragment on the extreme left is ˜20 kb and not shown toscale.

[0071]FIG. 2: Visualization of HA capsules in transformed bacteria bylight microscopy. These photomicrographs of early log cultures stainedwith India ink (Collins and Lyne, 1976) were taken on a Leitz Laboruxmicroscope at 1000× magnification. The results depict the ability ofplasmids pPD41 or pPD41Δ5, but not pAT19 alone or pPD4Δ7, to direct HAcapsule biosynthesis after transformation into the acapsular S. pyogenesmutant S43Tn7 or into normally acapsular E. faecalis. The bright halosurrounding the cells is the HA capsule. Ovine testicular hyaluronidasetreatment destroyed the capsule. Panels: A, wild-type S43; B,S43Tn7(pAT19); C, S43Tn7(pPD41); D, E. faecalis(pPD41Δ5); E, E.faecalis(pPD41Δ7); F, E. faecalis (pPD41Δ5) treated with 500 units/mlhyaluronidase (type V, 2000 u/mg) for 30 min at 37° C.

[0072]FIG. 3: PAGE analysis of polysaccharides produced by varioustransformed bacteria. Authentic HA (std) and polysaccharides purifiedfrom cell cultures as described by DeAngelis, et al. (1993a) wereelectrophoresed on a 4% gel and stained with Alcian Blue. Strainswithout pPD41 or pPD41Δ5 do not produce HA. The majority of the polymerpopulation in each sample migrated similarly to high MW HA (lanes F,G).The bracket on the right marks the extent of staining of the low MW HAstandard, which did not photograph well (lane H). The arrowheadindicates the top of the gel. Streptomyces hyaluronate lyase [HAase]treatment (20 units, 15 min) completely degraded the bacterial products.One μg (by carbazole assay) samples were loaded in lanes A-H and 8 μgsamples were loaded in I-K. Lanes: A, S43; B, S43Tn7(pAT19); C,S43Tn7(pPD41); D, E. faecalis(pPD41Δ7); E, E. faecalis(pPD41Δ5); F,native HA, viscosity=13,172; G, HA with viscosity=1,589; H, HA withviscosity=20; I, HAase-treated sample C; J, HAase-treated sample E; K,HAase-treated sample F.

[0073]FIG. 4: SDS-PAGE analysis of proteins synthesized bv pPD41deletion plasmids in E. coli minicells. Minicells labeled with[³⁵S]Met/Cys were lysed by boiling in SDS-sample buffer andelectrophoresed on a 10% gel. Cells containing the pPD41Δ5 plasmidproduce HA and two proteins are seen on this autoradiogram (24 hrexposure) at 42 and 45 kDa (lane 1, positions marked with arrows) thatare not produced by vector alone (lane 3). Cells containing the pPD41Δ7plasmid do not produce HA and only synthesize the 45 kDa protein (lane2). Standards (BioRad, low MW) are shown in kDa.

[0074]FIG. 5: Tn Mapping Analysis of Mutant and Transductant Strains.Southern analysis of HindIII digests of chromosomal DNA of various S43strains using a Tn-specific probe (³²P-panel, 48 hr autoradiogram)reveals that S43Tn7 (T) contains two Tn insertions (each Tn yields twobands due to an internal HindIII site). Transduction segregates the twoTns and produces nonmucoid (N,N′) or mucoid (M,M′) colonies (twoindependent clones of each are shown). Wild-type S43 (W) DNA does nothybridize with the probe. All the wild-type HindIII fragments detectedwith ethidium bromide (EB panel) migrate as ≦10 kb (S; λ HindIIIstandards in kb). Therefore, the chimeric Tn-tagged fragments (markedwith arrows) were purified and sequenced directly. An oligonucleotideprobe specific for the HA biosynthetic locus was derived from thefragment marked with the star.

[0075]FIG. 6: Schematic Map of the HA Biosynthesis Locus and VariousPlasmid Constructs. A restriction map of the complementing region of S43DNA, containing two substantial ORFs, is shown. The hasA and hasB genesare translated in the same orientation but in different reading frames.In this schematic, the HasA open reading frame begins with the standardATG codon. Sites for EcoRI (E), HindIII (H), ClaI (C), BglII (B), PstI(P), and EcoRV (R) are marked. The Tn insertion site was about 4 kb tothe right of the E site on the wild-type map but the interveningchromosomal DNA was deleted in the S43Tn7 mutant (DeAngelis, et al.,1993). The various pPD41 deletion constructs are depicted (black lines)below the map. The cross-hatched areas represent flanking sequences oneither side of the two open reading frames.

[0076]FIG. 7: Nucleotide and Deduced Protein Sequence of the HA Synthasegene, hasA. The DNA sequence surrounding the HA synthase ORF wasdetermined on both strands with Sequenase. The standard deduced startcodon for a protein (ATG) is indicated as the first amino acid in thisfigure. This putative start codon (ATG) is marked as position +1.Alternate start codons (Gren, 1984) indicated in bold-face (GTG at −72or TTG at −27 and −15) are present in-frame upstream from this ATG. Theadditional amino acids comprising HasA, if alternative start codons areused, are shown in lower case. Hydrophobic stretches predicted to bemembrane-associated are underlined and Cys residues are shown stippled.The beginning of HasB (Dougherty and van de Rijn, 1993) is also depictedat the lower right. The sequence is in the GenBank database underAccession No. L20853.

[0077]FIG. 8: E. coli Minicell Analysis of pPD41 Deletion Constructs.Minicells from χ1448 containing various plasmids were ³⁵S-labeled andthe proteins were separated on a 12.5% SDS-PAGE gel. This autoradiogram(10 hr exposure) shows that when hasA or hasB genes are disrupted, thepredicted proteins (HasA, filled arrow at 42 kDa; HasP, open arrow at 45kDa) are likewise affected. The truncated versions of HasA (filledcircle) or HasB (open circle) are smaller as expected. Lanes: A, pAT19vector, B, pPD41Δ7; C pPD41Δ5; D, pPDΔPstI; E, pPDΔEcoRV. Standards(BioRad, low MW) are marked in kDa.

[0078]FIG. 9: Buoyant density separation of acapsular and encapsulatedstreptococci. The procedure described herein in Example III was used inthis example to distinguish encapsulated, mucoid S43 cells andacapsular, nonmucoid NZ131 cells except that, for purposes ofillustration, the 65% Percoll layer was underlaid with a 100% Percollpad. Note that cells with a capsule (C) are at the top interface whilethe acapsular or enzyme-treated cells (A) appear at the lower interface(these cells would be in a pellet if not for the 100% Percoll pad).Hyaluronidase (HAase) treatment of the culture of the mucoid strainremoves the capsule and increases the density so that the majority ofthe cells appear in the pellet.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0079] The present invention will be exemplified herein in terms ofpreferred embodiments for the isolation and use of DNA segmentscomprising sequences encoding the HA synthase gene from Streptococcalsources. However, it will be appreciated by those of skill in the artthat in light of the present disclosure the invention is also applicableto the isolation and use of the HA synthase enzyme from virtually anysource, such as Streptococcus pyogenes, S. equisimilis, other group A orgroup C streptococcal strains or eukaryotic sources such as dermal orsynovial fibroblasts, chondrocytes, trabecular-meshwork cells or roostercomb mesodermal cells which contain HA synthase encoding DNA that isactively transcribed (and is a suitable source of mRNA for thepreparation of cDNA libraries).

[0080] The preferred application of the present invention to theisolation and use of streptococcal HA synthase DNA involves generallythe steps of (Sambrook, et al., 1989) isolation of streptococcal genomicDNA; preparation of a genomic DNA library, such as in a bacteriophagelambda; screening the library with oligonucleotides from the derivedsequence; isolating clones and subclones of phage with theoligonucleotide; excising the resident plasmid from within the phagegenome or ligating a purified DNA into a selected site in a cloningvector; (Trieu-Cuot, et al., 1991) transfection of host Streptococcus orE. coli cells with the recombined vector; and selection of coloniesexpressing HA synthase or HA itself through the application of speciallydesigned screening protocols. Following identification of a clone whichcontains the HA synthase gene, one may desire to reengineer the HAsynthase gene into a preferred host/vector/promoter system for enhancedproduction of HA.

[0081] A. Cloning of Hyaluronate Synthase Gene

[0082] To clone the HA synthase gene, hasA, the present inventors usedtransposon insertion mutagenesis to identify the locus that isresponsible for capsular formation. Genomic DNA from a transposon-taggedmutant of the Streptococcus pyogenes strain S43 was isolated frombacteria following hyaluronidase treatment, chloroform/isoamylextraction and ethanol precipitation. It is believed to be important toprovide a DNA fragment encoding a full length or essentially full lengthenzyme because the initial screening protocol requires expression of afunctional enzyme shown to synthesize HA.

[0083] The identification and verification of the HA synthase gene wasaccomplished by analyzing transposon-directed mutants and cells thathave been transformed with the HA synthase gene of the presentinvention. The assay is based on the formation of a polysaccharidecapsule in acapsular strains of Streptococci and heterologous bacteria.Initially TN916 insertional mutants were screened for an acapsular,non-mucoid phenotype. The ends of the transposon were then used toobtain sequence in the vicinity of the interrupted gene to direct thecloning of the wild-type hasA gene.

[0084] A contiguous three kilobase pair region of DNA (FIG. 1) wasisolated from Group A Streptococcus pyogenes [GAS] that can directhyaluronic acid [HA] capsule biosynthesis in acapsular mutants as wellas heterologous bacteria (FIG. 2). The DNA was identified by transposon916 insertional mutagenesis and subcloned into a plasmid shuttle vector.Mutant acapsular GAS or Enterococcus faecalis containing this plasmid,but not vector alone, displayed a mucoid phenotype on agar plates,possessed a capsule as seen by light microscopy, and produced HA inquantities comparable to wild-type GAS. The polysaccharide was shown tobe authentic HA based on its recognition by a specific HA-bindingproteoglycan and its degradation by Streptomyces hyaluronate lyase.Escherichia coli with the complementing plasmid also produced HA, but atonly 10% of the level made by the above cells. E. coli minicell analysisshowed that two proteins, 42 and 45 kDa, are expressed by the functionalDNA insert. Deletion analysis of the insert in the minicells revealedthat the 42 kDa protein is essential for HA production.

[0085] One may also desire to characterize the streptococcal or other HAsynthases in terms of their kinetics and physical and chemicalproperties. The parameters, K_(m) and V_(max), are determined from adouble reciprocal plot of the velocity of the reaction versus substrateconcentration (Lineweaver-Burke plot). Properties which may be ofinterest may include the enzyme's thermostability, optimum pH foractivity, effects of various ions, and effects of various inhibitors.Isoelectric focusing will be used to determine the isoelectric point ofthe synthase. Understanding these factors would provide basicinformation that may further allow one the ability to better determinewhat alterations in their primary sequence can provide additionaladvantages.

[0086] By appropriate modification of the DNA segment comprising thegene for HA synthase (e.g., deletion of membrane spanning domains of theprotein), the enzyme can be converted to a form that may be secreted bythe transfected bacterial host. This enzyme in soluble form, if stillactive in the ability to synthesize HA, would provide substantialimprovement in the ease of purification of this modified enzyme and inits potential utility in an enzyme reactor system for the in vitroproduction of HA.

[0087] B. Typical Genetic Engineering Methods Which May be Employed

[0088] If cells without formidable cell membrane barriers are used ashost cells, transfection is carried out by the calcium phosphateprecipitation method, well known to those of skill in the art (Sambrook,et al., 1989). However, other methods may also be used for introducingDNA into cells such as by nuclear injection, electroporation, protoplastfusion or by the Biolistic(tm) Bioparticle delivery System recentlydeveloped by DuPont (1989). The advantage of using this system is a hightransformation efficiency. If prokaryotic cells or cells which containsubstantial cell wall constructions are used, the preferred method oftransfection is calcium treatment using calcium chloride (Sambrook, etal., 1989) or electroporation.

[0089] Construction of suitable vectors containing the desired codingand control sequences employ standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and relegated in theform desired to construct the plasmids required. Cleavage is performedby treating with restriction enzyme (or enzymes) in suitable buffer. Ingeneral, about 1 μg plasmid or DNA fragments are used with about 1 unitof enzyme in about 20 μl of buffer solution. (Appropriate buffers andsubstrate amounts for particular restriction enzymes are specified bythe manufacturer.) Incubation times of about 1 hour at 37 degree(s) areworkable.

[0090] After incubations, protein is removed by extraction with phenoland chloroform, and the nucleic acid is recovered from the aqueousfraction by precipitation with ethanol. If blunt ends are required, thepreparation is treated for 15 minutes at 15 degree(s) C. with 10 unitsof Polymerase I (Klenow), phenol-chloroform extracted, and ethanolprecipitated. For ligation approximately equimolar amounts of thedesired components, suitably end tailored to provide correct matchingare treated with about 10 units T4 DNA ligase per 0.5 μg DNA. Whencleaved vectors are used as components, it may be useful to preventrelegation of the cleaved vector by pretreatment with bacterial alkalinephosphatase.

[0091] For analysis to confirm functional sequences in plasmidsconstructed, the ligation mixtures are used to transform E. coli K5strain Bi8337-41 (Gupta, et al., 1982), and successful transformantsselected by erythromycin resistance where appropriate. Plasmids from thelibrary of transformants are then screened for bacterial colonies thatexhibit HA production. These colonies are picked, amplified and theplasmids purified and analyzed by restriction mapping. The plasmidsshowing indications of a functional HA synthase gene are then furthercharacterized by sequence analysis by the method of Sanger (Sanger, etal., 1977), Messing (Messing, et al., 1981), or by the method of Maxam(Maxam, et al., 1980). ps C. Host Cell Cultures and Vectors

[0092] In general, prokaryotes are preferred for the initial cloning ofDNA sequences and construction of the vectors useful in the invention.It is anticipated that the best host cells may be Gram-positive cells,particularly those derived from the group A and group C Streptococcalstrains. Bacteria with a single membrane, but a thick cell wall such asStaphylococci and Streptococci are Gram-positive. Gram-negative bacteriasuch as E. coli contain two discrete membranes rather than onesurrounding the cell. Gram-negative organisms tend to have thinner cellwalls. The single membrane of the Gram-positive organisms is analogousto the inner plasma membrane of Gram-negative bacteria. The preferredhost cells are Streptococcus strains that are mutated to becomehyaluronidase negative or otherwise inhibited (EP144019, EP266578,EP244757). Streptococcus strains that have been particularly useful assuitable hosts include S. pyogenes S43, S. equisimilis and S.zooepidemicus.

[0093] Although E. coli is Gram-negative it is, nonetheless, a usefulhost cell in many situations, as shown in Examples I and IV. E. coliSURE™ cells were chosen as the initial recipient strain fortransformation and cloning of the HA synthase gene because this strainhas proven to be very useful in recombinant DNA studies. It is a widelyused host and is specifically engineered for recombinant DNA work. E.coli χ1448 was chosen for verification of HasA protein expressionbecause of its utility as a minicell expression system. Other E. colistrains may also be useful for expression of the shuttle vectors pAT19and pSA3 containing the HA synthase gene. For example, E. coli K12strain 294 (ATCC No. 31446) may be useful. Other strains which may beused include E. coli B, and E. coli K5. These examples are, of course,intended to be illustrative rather than limiting.

[0094] Prokaryotes may also be used for expression. For the expressionof HA synthase in a form most likely to accommodate high molecularweight HA synthesis, one may desire to employ Streptococcus species suchas S. equisimilis, S. pyogenes or S. zooepidemicus. The aforementionedstrains, as well as E. coli W3110 (F-, lambda-, prototrophic, ATCC No.273325), bacilli such as Bacillus subtilis, or other enterobacteriaceaesuch as Salmonella typhimurium or Serratia marcescens, and variousPseudomonas species may also be used, as described in Example V.

[0095] In general, plasmid vectors containing origins of replication andcontrol sequences which are derived from species compatible with thehost cell are used in connection with these hosts. The vector ordinarilycarries an origin of replication, as well as marking sequences which arecapable of providing phenotypic selection in transformed cells. Forexample, E. coli is typically transformed using pBR322, a plasmidderived from an E. coli species. pBR322 contains genes for ampicillinand tetracycline resistance and thus provides easy means for identifyingtransformed cells. A pBR plasmid or a pUC plasmid, or other microbialplasmid or phage must also contain, or be modified to contain, promoterswhich can be used by the microbial organism for expression of its ownproteins.

[0096] Those promoters most commonly used in recombinant DNAconstruction include the lacZ promoter, tac promoter, the T7bacteriophage promoter, β-lactamase (penicillinase) and tryptophan (trp)promoter system (Ausbel, et al., 1987). While these are the mostcommonly used, other microbial promoters have been discovered andutilized, and details concerning their nucleotide sequences have beenpublished, enabling a skilled worker to ligate them functionally withplasmid vectors (Ausbel, et al., 1987). Also for use with the presentinvention one may utilize integration vectors.

[0097] In addition to prokaryotes, eukaryotic microbes, such as yeastcultures may also be used. Saccharomyces cerevisiae, or common baker'syeast is the most commonly used among eukaryotic microorganisms,although a number of other strains are commonly available. Forexpression in Saccharomyces, the plasmid YRp7, for example, is commonlyused (Ausbel, et al. 1987). This plasmid already contains the trpl genewhich provides a selection marker for a mutant strain of yeast lackingthe ability to grow without tryptophan, for example ATCC No. 44076 orPEP4-1 (Jones, 1977). The presence of the trp1 lesion as acharacteristic of the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Suitable promoting sequences in yeast vectors include thepromoters for 3-phosphoglycerate kinase or other glycolytic enzymes,such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase.

[0098] In constructing suitable expression plasmids, the terminationsequences associated with these genes are also ligated into theexpression vector 3′ of the sequence desired to be expressed to providepolyadenylation of the mRNA and termination. Other promoters, which havethe additional advantage of transcription controlled by growthconditions are the promoter region for alcohol dehydrogenase 2,cytochrome C, acid phosphatase, degradative enzymes associated withnitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphatedehydrogenase, and enzymes responsible for maltose and galactoseutilization. Any plasmid vector containing a yeast-compatible promoter,origin of replication and termination sequences is suitable.

[0099] In addition to microorganisms, cultures of cells derived frommulticellular organisms may also be used as hosts. In principle, anysuch cell culture is workable, whether from vertebrate or invertebrateculture. However, interest has been greatest in vertebrate cells, andpropagation of vertebrate cells in culture (Tissue Culture, 1973) hasbecome a routine procedure in recent years (Sambrook, et al., 1989).Examples of such useful host cell lines are VERO and HeLa cells, Chinesehamster ovary (CHO) cell lines, and WI38, BHK, COS, and MDCK cell lines.

[0100] Other particularly useful host cell lines may be derived fromdermal or synovial fibroblasts, mesodermal cells of rooster comb or thetrabecular-meshwork cells of the eye. Expression vectors for such cellsordinarily include (if necessary) an origin of replication, a promoterlocated at the 5′ end of the gene to be expressed, along with anynecessary ribosome binding sites, RNA splice sites, polyadenylationsite, and transcriptional terminator sequences.

[0101] For use in mammalian cells, the control functions on theexpression vectors are often provided by viral material. For example,commonly used promoters are derived from polyoma, Adenovirus 2, bovinepapilloma virus and most frequently Simian Virus 40 (SV40). The earlyand late promoters of SV40 virus are particularly useful because bothare obtained easily from the virus as a fragment which also contains theSV40 viral origin of replication. Smaller or larger SV40 fragments mayalso be used, provided there is included the approximately 250 bpsequence extending from the HindIII site toward the BglI site located inthe viral origin of replication.

[0102] Further, it is also possible, and often desirable, to utilizepromoter or control sequences normally associated with the desired genesequence, provided such control sequences are. compatible with the hostcell systems. An origin of replication may be provided either byconstruction of the vector to include an exogenous origin, such as maybe derived from SV40 or other viral (e.g., Polyoma, Adeno, BPV) source,or may be provided by the host cell chromosomal replication mechanism.If the vector is integrated into the host cell chromosome, the lattermechanism is often sufficient.

[0103] Even though the invention has been described with a certaindegree of particularity, it is evident that many alternatives,modifications, and variations will be apparent to those skilled in theart in light of the foregoing disclosure. Accordingly, it is intendedthat all such alternatives, modifications, and variations which fallwithin the spirit and the scope of the invention be embraced by thedefined claims.

[0104] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

EXAMPLE I Isolation of the HA Synthase Locus

[0105] Tn insertional mutagenesis was used to tag and to mutate capsulebiosynthesis genes of GAS. The bacteriophage A25 transducing lysate fromone acapsular mutant, designated S43Tn7 and containing two Tn916elements, was found to transmit the nonmucoid phenotype to 3 out of 5transductants. The two Tn elements were segregated by transduction; oneTn insertion characterized by higher MW HindIII fragments was found onlyin the nonmucoid transductants (S43Td7N), while the other Tn insertionevent producing lower MW fragments was found only in the mucoid cells(S43Td7M). Nonmucoid transductants did not possess HA synthase activityor a capsule as determined by enzyme assays of membranes or by lightmicroscopy, respectively.

[0106] Materials and Methods

[0107] Materials: Media reagents were from Difco. Restriction and DNAmodifying enzymes were from Promega unless otherwise noted. Syntheticoligonucleotides were made at the UTMB Synthesis Facility or by KeystoneLaboratories. All other reagents were of the highest grade available andfrom Sigma except where stated otherwise.

[0108] Strains and Vectors: Escherichia coli was maintained on LB andgrown in Superbroth with antibiotics for plasmid production. Otherbacteria were grown as standing cultures on Todd-Hewitt brothsupplemented with 1% yeast [THY] and horse serum (5-10%, Gibco).Cultures to be assayed for HA were grown using the dialyzate fromdialyzed THY broth, (i.e. nutrients <10-14 kDa). The mucoid GAS strain,S43/192/4, was obtained from the Rockefeller Collection (Dochez, et al.,1919). Spontaneous strepr strains used as Tn acceptors were selected byplating ˜10⁹ cells on THY plates containing 1 mg/ml streptomycin and 3%defibrinated sheep blood (Colorado Serum). Enterococcus faecalis CG110,a Tn916 donor (tetracycline resistant, 5 μg/ml), and pAM118, a plasmidwith a Tn916 insert, were generously supplied by D. Clewell(Gawron-Burke and Clewell, 1984). The E. coli/Gram-positive shuttlevector pAT19 (erythromycin resistant, 8 μg/ml for Gram-positive or150-200 μg/ml for E. coli) was provided by P. Courvalin (Trieu-Cuot, etal., 1991). The E. faecalis host strain OG1RF was obtained from G. Dunny(Dunny, et al., 1991). E. coli minicell strain χ1448 (Meagher, et al.,1977) was supplied by R. M. Macnab. The E. coli hosts used were SURE,XL1-Blue (Stratagene), LE392, and KW251 (Promega).

[0109] DNA Purification and Sequencing: Streptococcal chromosomal DNAwas obtained by the method of Caparon and Scott (Caparon and Scott,1991). E. coli plasmid DNA was purified by the Instaprep method (5Prime→3 Prime, Inc.) for screening or by the SDS/alkali method forcloning and blotting procedures (Sambrook, et al., 1989). Agarose(BioRad) gel-isolated DNA <7 kb was purified by GeneClean (Bio 101),while longer fragments were isolated using GlassMax cartridges (Gibco)to minimize shearing. λ DNA was prepared from phage purified on glycerolgradients (Sambrook, et al., 1989). Sequencing of double strandedplasmids was performed with Sequenase 2.0 (US Biochem) andα-[³⁵S]thiodATP (Amersham).

[0110] Lambda Library Production: The λZAPII library (Stratagene, 1-10kb capacity) contained S43 wild-type DNA digested extensively withEcoRI. The λGEM system inserts (Promega, 9-23 kb capacity) consisted ofS43 DNA partially digested with Sau3A. The λZAP system inserts fromselected phage were excised and converted to plasmid form by coinfectionwith M13 helper phage (R408 or Exassist) according to Stratageneprotocols.

[0111] Transposon Mutagenesis and Mutant Selection: The detailed methodsfor Tn mutagenesis, mutant selection, and the isolation andcharacterization of the Tn-tagged DNA are described in Example 3.Briefly, Tn insertional mutagenesis was done on a mucoid strep^(r) S43strain using the method of O'Connor and Cleary (O'Connor and Cleary,1987). The nonmucoid mutant cells were enriched by Percoll stepgradients in analogy to work done with Group B Streptococci (H{dot over(a)}kansson and Holm, 1986) after overnight outgrowth in doubleselective media as in Example III and FIG. 9. Candidate mutants ofcapsule biosynthesis were picked by visually screening for dry, discretecolonies versus wild-type wet, spreading colonies. The nonmucoid strainschosen for study did not: a) float in 50% Percoll, b) produce a capsulevisualizable by India ink exclusion by light microscopy (Collins andLyne, 1976), c) possess detectable HA synthase activity in membranepreparations, or d) synthesize extracellular HA as determined by asensitive HA assay (see HA Polysaccharide Analysis). Transduction withthe streptococcal phage A25 (kindly supplied by M. Caparon) was used todetermine the relevance of the various Tn916 insertions in the nonmucoidstrains (Caparon and Scott, 1991).

[0112] Transposon Mapping and Isolation of the HA Synthesis Locus: Anoverview of the isolation procedures, as described herein below inExample 3, and as follows. After electrophoresis of chromosomal HindIIIdigests, the agarose gels (0.5-0.6%) were dried down directly (Ehteshamand Hasnain, 1991) and probed with the Tn916-containing EcoRI fragmentof pAM118 labeled by the random primer method (Sambrook, et al., 1989).It was noted that one of the Tn916/S43 chimeric DNA fragmentsconsistently migrated slower than the other fragments in HindIII digestsof wild-type DNA. This chimeric fragment from preparative digests (5-15μg) was isolated from an agarose gel with GlassMax and used as a DNAsequencing template to determine the sequence of the junction at thesite of insertion; the DNA was not cloned first. A syntheticoligonucleotide derived from the termini of the right HindIIIfragment-of Tn916 (Clewell, et al., 1988) that reads outward from the Tn(AAAGTGTGATAAGTCC)(SEQ ID NO:4) was employed as a primer in amodification of the Sequenase method (US Biochem) for plasmids. Theintact wild-type DNA was then obtained by screening the lambda librariesin the typical fashion (Sambrook, et al., 1989) with end-labeledoligonucleotide (TGGCACAATATGTCAGCCC)(SEQ ID NO:5) derived from thechromosomal sequence determined as above.

[0113] The HA biosynthesis locus is very unstable with respect to DNAdeletion. The present inventors found that this characteristic made thesubcloning difficult. The inventors found that three factors wereessential in order to obtain stable clones of the hasA locus in itsentirety: (1) use of a recombination deficient host (e.g. E. coli SURE™cells), (2) use of the electroporation method of transformation and, (3)performing the recovery and all further growth of the recombinant cellsat 30°-32° C. If these conditions were not followed for the subcloningof the hasA DNA, the vast majority of the target insert DNA was lost.Additionally, during the routine subculture of pPD41Δ5 at 37° C., theinventors have noted that deleted plasmids arose at a high rate.Therefore, a temperature of about 30° C. was used for any applicationsin which hasA and plasmid integrity was concerned.

[0114] To create a deletion set, the pPD41 plasmid linearized with XbaIand SphI was truncated by limited Exonuclease III digestion and MungBean nuclease treatment according to the Stratagene kit. The ligationmixtures were transformed into Epicurean SURE cells (Stratagene) andscreened for insert size. E. coli was also transformed by the Ca²⁺method (Sambrook, et al., 1989).

[0115] Results

[0116] The gel-purified, Tn-tagged chromosomal DNA from S43Tn7 was useddirectly as a template in sequencing reactions with a Tn-specific primerthat reads outward from the Tn terminus and into the insertion site. Anoligonucleotide probe corresponding to a portion of the sequence of theinterrupted DNA associated with the nonmucoid phenotype was then used asa hybridization probe for screening wild-type S43 genomic DNA librariesin λ phage. An excised λZAP clone, designated pB3, containing a 5.5 kbEcoRI fragment was obtained (FIG. 1). However, differences between thewild-type and Tn-mutant genomes were noted by Southern analysis. Sinceprevious studies showed that Tn916 can cause deletions of chromosomalDNA (Dougherty and van de Rijn, 1992), the inventors then determinedthat at least 4 kb of DNA flanking the small arm (5 kb HindIII portionof Tn) was missing in S43Tn7 and S43Td7N (FIG. 1). Therefore, a largerwild-type genomic fragment spanning the deletion was obtained from theλGEM library. A 6.6 kb portion of DNA adjacent to pB3 was subcloned intopAT19 and designated pPD41 (FIG. 1).

[0117] The various plasmids were introduced into acapsular Streptococcusmutants as well as into heterologous bacteria. Table II shows HAproduced by cultures (pooled spent media plus SDS cell extract) grown indTHY. HA was determined by the HA TEST assay kit (Pharmacia, Uppsala,Sweden). The HA concentration was normalized per A₆₀₀ unit of cells. ThepPD41 plasmid confers the ability to synthesize HA in the three speciestested (DeAngelis, et al., 1993a). The truncated version, pPD41Δ5, wasthe minimal size functional plasmid obtained. TABLE II HA production byvarious constructs. BACTERIAL HA STRAIN PLASMID ng/μl dTHY media alone —  1^(a) S. pyrogenes S43 — 1120 S43Tn7 pAT19   8 S43Tn7 pPd41  640S43Yn11^(b) pAT19   3 S43Tn11 pPD41  860 S43Tn11 pB3   6 E. faecalisOG1RF pAT19   2 pPD41  690 E. coli SURE pPD41  60 pPD41Δ4  80 pPD41Δ5 80 pPD41Δ6   2

EXAMPLE II Characterization of the HA Synthase Locus

[0118] When pPD41 was electroporated into the original acapsular Tnmutant, S43Tn7, or a spontaneously arising nonmucoid strain, S43Tn11,transformant colonies displayed the mucoid phenotype on agar plates,while cells with pB3 or pAT19 were nonmucoid. The capsules of thecomplemented cells were easily visualized by microscopy with India inkand were indistinguishable from the wild-type (FIG. 2). Ovine testicularhyaluronidase treatment of the cultures completely destroyed thesecapsules.

[0119] Using a sensitive radiometric assay, HA was detected in thecultures of the Tn-mutants containing pPD41 in amounts comparable to thewild-type parent (as shown in Table II). Transformants with pB3 orpAT19, as well as the original mutant without plasmid, did not produceHA (Table II). The HA was detected by proteoglycan binding; this highaffinity interaction is very specific and is widely accepted as evidencefor the presence of HA (Tengblad, 1980). As in the case of the GASmutants, E. faecalis or E. coli containing pPD41 produced HA (Table 1).By microscopy with India ink, E. faecalis, but not E. coli, containingpPD41 possessed a substantial capsule.

[0120] Materials and Methods

[0121] HA Synthase Preparation and Assay: HA synthase was obtained frommembranes of late log phase cells disrupted by sonication in PBS (20%cell suspension, dry ice/50% methanol bath, 4×2 min, Heat Systems W-380with microprobe). Debris was removed by centrifugation at 12,000 g×10min at 4° C., and then the membrane fraction was harvested from thesupernatant by ultracentrifugation (100,000 g×60 min). The membranes(5-300 μg protein) were incubated with UDP-[¹⁴C]GlcUA (250 mCi/mMole,ICN) in the assay as described by Triscott and van de Rijn (Triscott andvan de Rijn, 1986). Specificity of polymerization was tested by omittingUDP-GlcNAc. Incorporation of the radiolabel into high MW polymers wasmeasured by paper chromatography (Prehm, 1983).

[0122] HA Polysaccharide Analysis: The presence of HA in bacterialcultures (early log for S43 derivatives, late log for all others) wasdetermined using the HA TEST radiometric assay (range 5-500 ng,Pharmacia). The detection is based on inhibition of ¹²⁵I-proteoglycanbinding to HA immobilized on beads by soluble HA in the sample. Secretedor released HA in cultures grown on dTHY was measured by assay of thesupernatant fraction after centrifugation (11,000 g×5 min).Cell-associated HA was determined by extracting the cell pellet in 1/10vol of PBS containing 0.01% SDS for 40 min at 37° C. The cells were thenremoved by centrifugation as above. The final SDS concentration in theHA assay never exceeded 0.001%.

[0123] HA was also purified by cetyltrimethylammonium bromide (CTAB)precipitation. The pooled media supernatant and cell extracts (treatedwith 0.1 mg/ml trypsin for 40 min at 37° C. followed by SDS extractionas above) from 25 ml cultures were adjusted to 0.3% CTAB and allowed tostand at 37° C. for 15 min. The precipitate was collected bycentrifugation (3,000 g×20 min at room temperature) and resuspended in0.7 ml of 2 M NaCl with gentle mixing for 40 min. The solubilizedfraction (clarified by high speed centrifugation, 11,000 g×5 min) wasthen precipitated by addition of 2 volumes of ethanol. After 5 min, thesolids were collected by high speed centrifugation. The pellet waswashed with 70% ethanol/30% 2 M NaCl and then 70% ethanol. After briefdrying, the pellet was resuspended in 2 M NaCl and ethanol precipitationwas repeated as above. The final pellet was dissolved in 0.2 ml waterovernight with gentle mixing at 4° C.

[0124] The uronic acid content of the purified material was measured bythe carbazole assay with glucuronic acid as standard (Bitter and Muir,1962). The MW of the polymers and authentic rooster comb HA standards(Lifecore) were compared by polyacrylamide gel electrophoresis (PAGE)(Min and Cowman, 1986) and size exclusion chromatography. To verify thenature of the polysaccharide, samples were digested with hyaluronatelyase from Streptomyces hyaluroniticus in 50 mM sodium acetate, pH 5.3,at 42° C. before analysis. Sepharose 4B (Pharmacia, 1×25 cm column, 20ml bed volume) eluted with PBS was used to fractionate the variouspolymers. The column fractions were assayed by the carbazole method andthe HA TEST kit was used to confirm the major peak identity. The columnwas calibrated with dextrans (2×10⁶, 5×10⁵, 4×10⁴ Da; Pharmacia) andlactose as well as the HA standards.

[0125] Minicell Analysis: The identity of plasmid-encoded polypeptideswas determined by radiolabeling proteins produced in minicells(Matsumura, et al., 1977). Minicells from E. coli χ1448 containingpAT19, pPD41Δ5, or pPD41Δ7 were harvested from sucrose gradients andwashed with PBS containing 0.01% gelatin. The. minicells were incubatedat 37° C. for 1 hr in minimal salts with glycerol and all amino acidsexcept Met and Cys. The minicells were then labeled with ³⁵S-Translabel(ICN) for 30 min at 37° C. followed by a 5 min Met/Cys chase. Theminicells were then washed with PBS and analyzed by SDS-PAGE afterboiling (3 min) in Laemmli sample buffer (Laemmli, 1970). The gels werestained with Coomassie Blue, dried, and exposed to XAR-5 film (Kodak).

[0126] Miscellaneous: Of several published electroporation methods foruse with Gram-positive bacteria, the present inventors found that onlythe technique of Caparon and Scott (Caparon and Scott, 1991 wassuccessful in transforming S43 derivatives with plasmids (0.5 to 20transformants/μg DNA).

[0127] Results

[0128] To determine the minimum size of the locus directing HAbiosynthesis, the complementing DNA insert of pPD41 was reduced bylimited exonuclease digestion of the plasmid from the SacI end of thepAT19 multiple cloning site. E. coli transformed with plasmidscontaining an insert of ˜3 kb (e.g. 5 min deletion, pPD41Δ5; see FIG. 1)still produced HA, while cells with smaller inserts (e.g. 6 mindeletion, pPD41Δ6, ˜2.3 kb or 7 min deletion, pPD41Δ7, ˜1.7 kb) did notmake HA (Table II). The E. faecalis cells transformed with pPD41Δ5produced hyaluronidase-sensitive capsules as assessed by microscopy(FIG. 2D & 2F), and formed mucoid colonies on agar plates, whereas thecells containing pPD41Δ7 were equivalent to untransformed E. faecalis(FIG. 2E). E. faecalis has not been reported to produce a capsule or anyexopolysaccharides. Therefore, the pPD41Δ5 insert is responsible for HAcapsule biosynthesis.

[0129] The extracellular polysaccharides produced by the variousbacteria containing the pPD41 family of plasmids were furthercharacterized by gel filtration chromatography and PAGE. Allpolysaccharides possessed M_(r)s on the order of 10⁶, since they elutedin the void volume and the first included fractions on the Sepharose 4Bcolumn, well before a 500 kDa dextran standard (not shown). Byelectrophoretic analysis, wild-type S43 HA and polysaccharide frommutant GAS strains with pPD41 or E. faecalis with pPD41Δ5 migratedsimilarly compared to authentic high MW HA standards (FIG. 3). Thespecific Streptomyces hyaluronate lyase degraded both authentic HA andthe polysaccharides produced by S43Tn7 or E. faecalis containing thecomplementing plasmids (FIG. 3).

[0130] The E. coli minicell system provides a convenient way todetermine the number and size of proteins encoded by genes on episomalplasmids (Meagher, et al., 1977; Matsumura, et al., 1977). Minicellanalysis revealed that at least two proteins were encoded on thecomplementing DNA that directed HA capsule biosynthesis (FIG. 4). Inaddition to vector-derived proteins, the nonfunctional pPD41Δ7 encoded aprominent 45-kDa protein. Minicells with the HA-producing pPD41Δ5plasmid produced a 42-kDa protein as well as the 45-kDa species,indicating that the former protein is essential for HA synthesis. Thepresent inventors calculate that about 80% of the coding capacity of the˜3 kb insert in pPD41Δ5 is utilized for these two proteins.

[0131] Neither the purification nor the cloning of the HA synthase hasbeen successfully demonstrated in either bacteria or eukaryotes. Prehmand Mausolf (Prehm and Mausolf, 1986) implicated a 52-kDa protein fromGCS as the HA synthase by affinity labeling with periodate-oxidizedsugar nucleotides. A polyclonal antibody to this protein inhibited HAsynthase activity of membranes (Prehm and Mausolf, 1986). However, theactive HA synthase was not purified. The gene corresponding to the 52kDa protein was then cloned using the antibody and, although theassertion of cloning the HA synthase gene was made, the presentinventors have found this conclusion to be invalid; the deduced sequencehad similarities to an oligopeptide transport protein of Bacillussubtilis (Lansing, et al., 1993). van de Rijn and Drake (van de Rijn andDrake, 1992) found three polypeptides (42, 33, and 27 kDa) from GAS andGCS membranes that were photoaffinity labeled by a substrate analogue,azido UDP-glucuronic acid. Excess UDP-GlcA inhibited incorporation ofthe analogue but labeling of all three polypeptides was stimulated uponaddition of the other precursor of HA, UDP-GlcNAc (van de Rijn andDrake, 1992). The 42 kDa protein labeled in the pPD41Δ5-containingminicells is the same size as one of the proteins photoaffinity labeledwith a substrate analog (van de Rijn and Drake, 1992). However, theproteins of 32 and 27 kDa that were also labeled were not observed inthe inventors studies. Dougherty and van de Rijn (Dougherty and van deRijn, 1992) used Tn insertional mutagenesis to identify a GAS geneticlocus associated with HA synthase activity. Two open reading frames weredescribed schematically but no sequence information was reported and noin vivo or in vitro HA synthase activity was reported.

[0132] None of the above studies functionally reconstituted HA synthesisin an acapsular mutant or in heterologous bacteria with clonedstreptococcal DNA. The results of the inventors, however, show that acontiguous 3 kb region of the GAS chromosome, encoding proteins of 42and 45 kDa, can direct HA biosynthesis in GAS mutants as well as in E.faecalis and Gram-negative E. coli.

EXAMPLE III Cloning of the HA Synthase Gene

[0133] The HA synthase gene of GAS was initially identified by Tninsertional mutagenesis as described in Example I. The bacteriophage A25transducing lysate (Caparon and Scott, 1991) from one acapsular mutant(designated S43Tn7), which contained two Tn elements, transmitted thenonmucoid phenotype to 3 out of 5 transductants (FIG. 5). The nonmucoidtransductants did not possess HA synthase activity or a capsule by lightmicroscopy, but the mucoid transductants were equivalent to wild-typeS43. HindIII digests of mutant S43Tn7 chromosomal DNA showed two bandsmigrating at 16 and 18 kb on agarose gels that corresponded to thehigher MW bands detected by a Tn-specific probe on Southern blots of allTN916 mutants (FIG. 5). These larger species are the result of adding 10kb of Tn DNA to the S43 HindIII fragment at the insertion site.

[0134] Since the Tn-tagged DNA from S43Tn7 was well resolved from theother HindIII fragments, it could be gel-purified. The 18 kb chimericfragment associated with the HA biosynthesis defect was therefore useddirectly as a template for sequencing reactions with a Tn-specificprimer that reads outward from the Tn terminus and into the interruptedgene. An oligonucleotide (SEQ ID NO:5), corresponding to a portion ofthe sequence of the interrupted gene from the 18 kb chimeric fragment,was used as a hybridization probe for screening wild-type S43 genomicDNA libraries in λphage.

[0135] An excised λZAP clone, pB3, containing a 5.5 kb EcoRI fragmentwas selected and studied further. However, Southern analysis utilizingvarious oligonucleotide probes to the sequence of pB3 revealed somediscrepancies between the wild-type and Tn-mutant genomes (e.g. the SEQID NO:6 oligo hybridized to S43 but not S43Tn7, while the SEQ ID NO: 5oligo hybridized to both) . Therefore, a larger genomic fragmentspanning the Tn-induced deletion (DeAngelis, et al., 1993a,b) wasobtained from the λGEM library. After an extensive subcloning effort andsubsequent exonuclease III deletion, a 3.2 kb fragment of S43 DNA wasidentified as a locus that could direct HA biosynthesis (DeAngelis, etal., 1993a).

[0136] Materials and Methods

[0137] Materials and Strains: Restriction and DNA modifying enzymes werefrom Promega unless otherwise noted. All other reagents were of thehighest grade available from Sigma unless stated otherwise. Mediareagents were from Difco. Cultures to be assayed for HA were grown usingthe dialysate from dialyzed THY broth (i.e. nutrients <10-14 kDa). Themucoid GAS strain, S43/192/4, was obtained from the RockefellerCollection (Dochez, et al., 1919). E. coli K5 (Bi8337-41) was obtainedfrom I. and F. Orskov (Copenhagen, Denmark; Gupta, et al., 1982). Allother strains and plasmids used were described by DeAngelis, et al.,(1993a).

[0138] Tn Mutagenesis and Mutant Selection: Tn insertional mutagenesiswas conducted by the method of O'Connor and Cleary (O'Connor and Cleary,1987) except that ovine hyaluronidase (Type V) was added to the GASculture (0.2 mg/ml, 1 hr at 37° C.) after overnight growth and used at ahigher concentration (0.1 mg/ml) in the mating plate media. The Tn916donor, Enterococcus faecalis CG110 (Gawron-Burke and Clewell, 1984), wasmated on nitrocellulose filters (88 mm, 0.45 μm, Micron Separation,Inc.) with strepr S43. The mating mixture was scraped off the filterswith 0.4 ml THY containing 1 mg/ml streptomycin and 5 μg/mltetracycline.

[0139] The nonmucoid mutant cells were then enriched over Percoll(Pharmacia) step gradients (DeAngelis, et al., 1993b; H{dot over(a)}kansson and Holm, 1986) as illustrated in FIG. 9. This selectionprocess allowed about a thousand-fold more bacteria to be more readilyexamined than if plating methods were used directly after the matingstep. Acapsular (or hyaluronidase-treated) cells pellet through 50%Percoll, but mucoid cells float at the interface. After overnightoutgrowth (50-70 μl mating mixture/5 ml double selective media with 5%serum in a 15 ml tube), the cultures were underlaid with 2 ml of 50%Percoll in water and centrifuged (3,000 g×10 min). The media, the cellsat the interface, and most of the Percoll were removed by aspiration andthe high density “cell pellet” fraction was then used to inoculate 5 mlof fresh double selective media. Two further rounds of outgrowth for 4-8hrs (A₆₀₀=0.2-0.6) and gradient enrichment were performed. Portions ofthe final cell pellet were streaked on double selective platescontaining 5% sheep blood and visually screened for candidate mutants ofcapsule biosynthesis: those with dry, discrete colonies versus wild-typewet, spreading colonies.

[0140] The mutants were streak-purified and verified to be similar towild-type S43 with respect to vigor, β-hemolysis, DNase secretion (usingDNA/methyl green agar), and production of Group A carbohydrate(Ventrescreen, Hycor). Thirteen strains did not have HA synthaseactivity, produce capsules or contain HA (DeAngelis, et al., 1993a) butonly one strain, S43Tn7, transduced (Caparon and Scott, 1991) thenonmucoid phenotype.

[0141] Tn Mapping and Gene Isolation: Chromosomal DNA purified (Caparonand Scott, 1991) from the mutants and transductants was cut with HindIIIand analyzed by Southern hybridization. After electrophoresis, theagarose gels were dried down directly (Ehtesham and Hasnain, 1991) andprobed with the Tn-containing EcoRI fragment of pAM118 (Gawron-Burke andClewell, 1984) labeled by random priming (New England Biolab kit). Thehybridization was conducted overnight at 65° C. in 1×HPB (0.5 M NaCl, 5mM EDTA, 0.1 M Na₂PO₄, pH 7.2) containing 1% sarcosyl, and the gel wasthen washed for 40 min with 20 mM Tris HCl, pH 8 at 22° C.

[0142] The 16 or 18 kb chimeric Tn-tagged fragments from preparativedigests (5-15 μg) of S43Tn7 were isolated from gel slices using GlassMAX(Life Technologies) according to the manufacturer's instructions exceptthat the DNA was eluted from the GlassMAX unit with 3 sequentialadditions of water at 65° C. The Sequenase method (USBiochem) forplasmids, with modifications noted below, was employed to sequence thejunction at the site of Tn insertion directly from chromosomal DNA. Asynthetic oligonucleotide (AAAGTGTGATAAGTCC)(SEQID NO:4) based on thetermini of the right arm of TN916 that reads outward into theinterrupted gene was used as the primer (Clewell, et al., 1988).

[0143] The purified DNA fragment (50-100 ng) was denatured with NaOH,neutralized with sodium acetate, and quickly ethanol precipitated in thepresence of 10 μAg of phenol/CH₃Cl extracted glycogen. The primer SEQ IDNO:4 (0.22 pmol) was annealed to the template by slow cooling from 65°to 30° C. The labeling phase of the reaction was done with Mn²⁺ buffer,1:15 diluted labeling mix, and α[³⁵S]thiodATP (Amersham, 3,000 Ci/mMol)for 2 min at 20° C. The termination phase was done for 5 min at 37° C.with extension mix in the A and T reactions (0.6 μl) due to the A/T-richnature of streptococcal DNA. Gels were electrophoresed, processed(Sambrook, et al., 1989), and exposed to XAR-5 film for 1-10 days atroom temperature. Typically, the sequence of the Tn terminus/junction(6-10 bp) and 20-40 bases of the adjacent tagged streptococcal DNA wereobtained.

[0144] The oligonucleotide derived from the chromosomal sequencedetermined above was used to screen two lambda libraries (DeAngelis, etal., 1993a,b) to obtain the intact wild-type DNA, in which the Tninsertion had occurred in the mutant. The phage were adsorbed ontonitrocellulose filters and processed in the typical fashion (Sambrook etal., 1989). The filters were hybridized with end-labeled oligonucleotide5′-TGGCACAATATGTCAGCCC-3′ (SEQ ID NO:5), in 1.8×HPB (1 pmol probe/8 ml)with 1% sarcosyl, 0.5% nonfat milk at 42° C. for 3 hr and washed with0.5×HPB at the same temperature for 1 hr. The plaques yielding thestrongest signal were replated and rescreened twice. Purified phage froma λZAP library were converted to plasmid form by coinfection of SURE orSOLR cells (Stratagene) with the Exassist helper phage (Stratagene). Oneclone, pB3, was analyzed by sequencing with Sequenase using the standardprotocols. The phage selected from the λGEM library using SEQ ID NO:5were screened with another oligonucleotide(5′-TATGGCTTAGTGCCATTCG-3′)(SEQ ID NO:6), corresponding to the sequencefound near the end of the pB3 insert, in order to obtain DNA adjacent topB3.

[0145] Two positively hybridizing XGEM isolates, which formed smallplaques and grew poorly in liquid lysates, were obtained. Large scaleplate lysates with top and bottom agarose were needed in order toprepare their DNA (Sambrook, et al., 1989). The two clones (λ1X and λ2Ywith 20 and 12 kb inserts, respectively) contained the same region ofDNA as determined by direct sequencing of the λ DNA insert using theCircumvent method (New England Biolabs) and end-labeled oligonucleotideSEQ ID NO:6. The sequence obtained beyond the EcoRI site of pB3 (leftsite; DeAngelis, et al., 1993a) was used to make another oligonucleotide(5′-CAATCATACCACCAACTGC-3′)(SEQ ID NO:7), for mapping analysis of the λclones treated with various restriction enzymes.

[0146] Southern blot analysis showed that a fragment of about 7 kb couldbe excised from the smaller λ2Y clone using the EcoRI site in the S43DNA and the SacI site of the λ vector. A portion of the digest waspurified with a Magic minicolumn (Promega) and the fragments wereligated to pAT19 shuttle vector (Trieu-Cuot, et al., 1991) digested withEcoRI and SstI (Life Technologies). Attempts to subclone thestreptococcal fragment in its entirety were thwarted by spontaneousdeletions upon transformation into E. coli JM109. After using Epicureancompetent SURE cells (Stratagene), using 32° C. for transformationrecovery and all further growth, and restriction mapping ˜70 recombinantcolonies, a clone containing a 6.6 kb insert, designated pPD41, wasobtained that could complement the HA biosynthesis defect of mutant GAS(DeAngelis, et al., 1993a).

[0147] Results

[0148] Isolation of Mutants

[0149] The inventors have used the difference in relative buoyantdensity to isolate acapsular mutants from a mating mixture ofStreptococcus pyogenes strain S43 and an Enterococcus faecalistransposon TN916 donor (DeAngelis et al., 1993b). After centrifugationover a simple Percoll step gradient (50% pads), the cell pellet washarvested and used as a culture inoculum. Repeated cycles of growth andseparation on Percoll gradients were performed to enrich for acapsularmutants in order to avoid painstakingly screening hundreds of platesinoculated with unselected cultures. The initial cell “pellet” may notbe visible, but if it is processed for 3 additional cycles (afteraspiration of the supernatant) even spontaneous acapsular mutants thatoccur at low frequency are obtained. Conversely, mucoid varieties can beenriched from mixtures of both cell-types or spontaneous revertants ofnonmucoid mutants can be recovered; in this case the interface isharvested with a pipette and repeatedly processed as above. Isolation ofeither phenotype is finally accomplished by streaking out on agarplates. Quantitation of the capsular and acapsular phenotypes in apopulation of bacteria may be obtained by measuring the ratio ofrelative A₆₀₀ of resuspended cells harvested from both locations in thestep gradient. This method is broadly applicable to other encapsulatedmicroorganisms besides Group A Streptococci (e.g. Group C; unpublishedobservation) but optimization of the Percoll concentration and capsuledegrading reaction conditions may be necessary.

[0150] The utility and optimization of the buoyant densitycentrifugation technique was also studied with various encapsulated andacapsular strains. The inventors have determined optimal concentrationsof Percoll for separating encapsulated cells and acapsular cells byfirst using discontinuous step gradients of Percoll (e.g. 50%, 63%, 75%,87%). Wild-type S43 cells were found at the 50%/medium interface.Hyaluronidase-treated wild-type S43 cells collected at the 63%/75%Percoll interface near the green marker beads (DMB-7, 1.10 mg/ml). Thisdensity value was close to the measured value reported as the “typical”bacterial cell density (1.08 mg/ml) in a recent survey (Guerrero, etal., 1985). Light microscopy with India ink (Collins and Lyne, 1976) wasused to examine bacteria at both positions after centrifugation; all thecells at the medium/50% interface possessed capsules, while the cellswith the highest density at the 63%/75% interface had no detectablecapsule.

[0151] For bacteria with smaller capsules than the highly mucoid S43strain, a simple 65% Percoll pad could distinguish capsule phenotypesamong an array of Group A streptococci strains. Nonencapsulated cellswere found in the pellet, while encapsulated cells appeared at theinterface between the yellow media and clear Percoll (FIG. 9). Theinventors determined the presence or absence of HA on the variousstrains by using a sensitive radiometric assay with a detection limit of0.4 μg/ml of culture media. If the bacteria made at least 7 μg of HA perml of cells per 1 A₆₀₀ unit, they were found at the interface of thePercoll layer and media. One strain, DW 1009, that produced 4 μg of HAper ml of cells, however, appeared in the pellet and, therefore, appearsto be below the limit of detection by our simple and rapidcentrifugation method. This strain, however, showed nonmucoid colonymorphology and no evidence of a capsule by light microscopy.Hyaluronidase-treatment of the cultures of mucoid strains beforecentrifugation caused the vast majority of cells to pellet (FIG. 9),although some small clumps of cells may remain at the medium/Percollinterface due to incomplete digestion of their capsule.

[0152] The density separation method is surprisingly sensitive to thebacterial HA level. Strain NSA156 produced 7 μg/ml HA and floated on 65%Percoll. This strain, which produces only about 0.5% to 4% of the HAmade by most encapsulated strains, does not appear mucoid on plates andits capsule was not visible with the light microscope. A further assetof the present method is that very small amounts of cells are readilyvisible; cells at the 65% Percoll/media interface, when viewed at anangle, cloud the junction's usual “mirror-like” appearance, while higherdensity cells are concentrated by the conical bottom of the centrifugetube. Another benefit of this method is that it circumvents the need forfresh (<2 month old) radiometric assay kits for detection of hyaluronicacid and, therefore, such kits do not need to be continuously availablein the laboratory. If further quantitation is needed on selectedstrains, these can also be stored and tested at a later date.

[0153] This density separation method is well suited for the sensitivedetermination of the presence of an HA capsule in clinical streptococciisolates; bacitracin-sensitive, β-hemolytic colonies from standard bloodplates can be picked and assayed. In light of the capsule's importanceas a virulence factor and the resurgence of streptococcal diseases inthe USA and Europe, monitoring HA capsule production may be useful fortracking virulent strains or epidemiological trends. The only equipmentneeded is a 37° C. incubator or waterbath and a low-speed clinicalcentrifuge. In less than one day after the initial streak isolation onan agar plate, and with only a few minutes of hands-on labor, manyisolates can be screened for capsule production. Inclusion of thehyaluronidase-treated control tube may not be necessary during routinescreening, but rather can be used subsequently for verification of HAproduction.

[0154] hasA Cloning

[0155] The sequence of the complementing streptococcal DNA, the insertof pPD41Δ5, was obtained using both the nested nuclease deletion setwith the M13 vector primers and the functional plasmid with customoligonucleotides. Two major ORFs were present (FIG. 6) in agreement withthe earlier minicell analysis (DeAngelis, et al., 1993a). The sequenceof the first ORF, hasA, reveals the primary structure of a previouslyundescribed protein (FIG. 7)(DeAngelis, et al. (1993b). The deducedpolypeptide contains 395 residues with a M_(r)=45,063 if the standardATG initiation codon is used (or up to 419 residues if the alternate GTGinitiation codon at position −72 is used). The 42-kDa protein observedby SDS-PAGE analysis of pPD41Δ5 minicells is assigned to be HasA becausethe pPD41Δ7 plasmid, missing about half of the hasA gene (FIG. 6), doesnot produce the 42 kDa species (FIG. 8). HasA is predicted (Kyte andDoolittle, 1982) to be an integral membrane protein due to at least fourmembrane-associated regions (3 predicted transmembrane segments) and tohave a pI of 8.2.

EXAMPLE IV Further Characterization of the hasA Gene

[0156] To identify the role of the two genes on the complementingstreptococcal DNA, two constructs were made that substantially truncatedeither hasA or hasB (FIG. 6). One plasmid, pPDΔEcoRV, should produce theintact 45-kDa protein, HasB. The other, pPDΔPstI, should make the intact42-kDa protein, HasA. The pPDΔEcoRV construct, in which the truncatedHasA gene produced a new 27-kDa species (instead of the 42-kDa protein)as determined in minicells (FIG. 8), did not confer the ability toproduce HA in any host (Table III).

[0157] Materials and Methods

[0158] Polypeptides encoded by plasmid genes were identified by³⁵S-labeling of proteins produced in minicells from E. coli χ1448(Meagher, et al., 1977), containing pAT19 alone or various constructscontaining S43 DNA, as described by DeAngelis (DeAngelis, et al.,1993a). DNA purification and sequencing, lambda library production,nested deletion set construction, and HA synthase preparation and assaywere performed as described earlier (DeAngelis, et al., 1993a). Targetedinternal deletions were made by digesting pPD41Δ5 DNA with either EcoRVor PstI, purifying the DNA with Magic minicolumns and recircularizing byligation. The ligation mixtures were transformed into ElectrocompetentSURE cells (Stratagene) and screened for insert size. HA was quantitatedusing the Pharmacia HA Test Kit (DeAngelis, et al., 1993a). UDP-Glcdehydrogenase activity was measured as described (Dougherty and van deRijn, 1993) except cells (from 4.5 ml overnight cultures in dialyzedTHY, washed and resuspended in 0.4 ml buffer) were disrupted byvortexing with an equal volume of washed glass beads (75-150 μm, 5×30 sat 4° C. with 30 s on ice between mixing). The extracts were assayed at30° C. for a UDP-Glc-dependent increase in A₃₄₀ corresponding to NADHproduction. Protein was measured by the Bradford assay (Bradford, 1976)with a BSA standard.

[0159] Results

[0160] Minicells containing pPDΔPstI produced two nonvector-derivedproteins, the intact 42-kDa protein and a 29-kDa truncated version ofHasB (FIG. 8). The deleted hasB gene product is predicted to be 23-kDabased on the sequence. When transformed into SURE or χ1448 cells,pPDΔPstI could not direct HA synthesis (Table III). On the other hand,E. coli K5 transformed with pPDΔPstI could produce HA (Table III). Thisobservation should be the result of the endogenous UDP-Glcdehydrogenase, which is responsible for producing UDP-GlcA needed for K5capsular polysaccharide synthesis, substituting for the nonfunctionalstreptococcal enzyme. To verify this, the present inventors assayedstrains with the various plasmid constructs for UDP-Glc dehydrogenaseactivity (Table III). Indeed, all K5 cultures, including those withvector pAT19 alone, demonstrated this activity. SURE or χ1448 cells withplasmids encoding an intact 45-kDa protein possessed elevated enzymeactivity, whereas cells with the pPDAPstI plasmid possessed levelssimilar to host cells alone. TABLE III HA Production and UDP-GlcDehydrogenase Activity in E. coli Strains Containing Various pPD41Constructs HA UDP-Glc DH PROTEIN^(a) STRAIN PLASMID ng/μl^(b)pmol/min/μg^(c) HasA HasB SURE 0 3 − − PD41Δ5 61 19 + + PD41Δ7 0 7 − +PD41ΔPstI 0.2 0.8 + − PD41ΔEcoRV 0 14 − + χ1448 AT19 0 0.8 − − PD41Δ5 2117 + + PD41Δ7 0 6 − + PD41ΔPstI 0.6 2.5 + − PD41ΔEcoRV 0 10 − + K5(Bi8337- AT19 0 12 − − 41) PD41Δ5 253 12 + + PD41Δ7 0 17 − + PD41ΔPstI49 13 + −

[0161] These above results demonstrate that the hasA gene product, HasA,is-the 42-kDa protein, and the HA synthase. The 45-kDa protein derivedfrom hasB, is the UDP-Glc dehydrogenase (Dougherty and van de Rijn,1993). Furthermore, studies confirm that the 42-kDa protein has bothUDP-GlcNAc and UDP-GlcA glycosyl transferase activities. Crude membranesfrom the various constructs show HA synthase activity only in cells withthe intact hasA gene. UDP-¹⁴C-GlcA or UDP-³H-GlcNAc are incorporatedinto hyaluronidase-sensitive product only in the presence of UDP-GlcNAcor UDP-GlcA, respectively. This incorporation is decreased by >98% ifUDP-GalNAc or UDP-Glc are substituted for UDP-GlcNAc or if UDP-Glc orUDP-GalA are substituted for UDP-GlcA.

[0162] Dougherty and van de Rijn (Dougherty and van de Rijn, 1993)proposed in their later model that three ORFs (hasA, hasB, and hasC) areinvolved in HA biosynthesis. The inventors found that the S43 strainHasB is 99.8% identical at the nucleotide level to their GAS strain HasBsequence; there was perfect conservation at the protein level (notshown). The region containing the hasA and hasB genes (Dougherty and vande Rijn, 1993) possesses a restriction map consistent with the two ORFsfound in pPD41Δ5 (FIG. 6). The inventors also found that a putative HasCgene is present in S43, but is not required for HA biosynthesis. Neitherthe HasB nor HasC proteins are needed when both sugar nucleotideprecursors are present.

[0163] HA synthase possesses significant homology with the nodC geneproduct of Rhizobium. NodC is a membrane enzyme that synthesizeschitin-like (poly-β-1,4-GlcNAc backbone) oligomers (Lerouge, et al.,1990) which is a very analogous activity to that of streptococcal HAsynthase. NodC possesses several stretches of residues that areidentical or similar to the HA synthase. Overall the two proteins are30.6% identical. The hydropathy plots of the two proteins are verycomparable, including three predicted transmembrane segments in the samelocation near the carboxyl terminus (not shown). Other proteins withhomology to HA synthase include DG42 from Xenopus laevis, yeast chitinsynthase II, and an associated protein CSH2 (Bulawa, 1992). The 52-kDaprotein described by Prehm and coworkers (Prehm and Mausolf, 1986;Lansing, et al., 1993) is not homologous to HasA or these otherproteins. The gene cloned by these workers is not the HA synthase gene.

[0164] hasA and hasB are the only exogenous genes required to direct HAbiosynthesis in most bacteria, due to the presence of one of the sugarnucleotide precursors of HA, UDP-GlcNAc, which is necessary for cellwall formation. In cells that make both UDP-GlcNAc and UDP-GlcA only HAsynthase, the gene product of hasA, HasA, is needed to polymerize the HApolysaccharide (DeAngelis, et al., 1993b).

EXAMPLE V Large Scale Production of Hyaluronic Acid

[0165] This example is directed to the engineering of a bacterium thatoverproduces and secretes large quantities of HA, which can then bepurified from the medium. An engineered organism that overproduces HAwill make it cheaper to produce larger quantities of HA than presentlypossible. Reduced HA production costs will increase the number and typeof commercially viable products that can be developed.

[0166] The present inventors have cloned and sequenced a 3,200 base pairStreptococcal DNA fragment that confers on recipient bacteria theability to make HA as described in Examples II, IV and V. Analysis ofthis locus revealed the presence of two tandem genes (FIG. 6).Transformation of mutant, capsule-deficient Streptococcus cells withthese two genes restored their ability to make HA. Most importantly,putting these two genes into very different bacteria such as Escherichiacoli or Enterococcus faecalis also allowed these bacteria to produce HA.This was shown visually by noting the presence of a new capsule of HAsurrounding the cells (FIG. 2), and biochemically by using a specificassay to detect and quantitate HA (Tables II and III). This resultindicates that after it receives the hasA and hasB genes, any type ofbacteria will be able to make HA.

[0167] The nucleotide sequence of this 3.2 kb Streptococcal DNA fragmentshowed that the hasB gene encodes the enzyme UDP-glucose dehydrogenase,which is required for the cell to make UDP-glucuronic acid (UDP-GlcA),one of the two sugar precursors needed for HA biosynthesis. The secondsugar precursor needed for HA synthesis is UDP-N-acetylglucosamine(UDP-GlcNAc), present in all bacteria and required for cell wallsynthesis. The dehydrogenase gene was also reported by others (Doughertyand van de Rijn, 1993).

[0168] The inventors have found that any cell containing a functionalUDP-Glc dehydrogenase and a functional HA synthase can make HA. Not allE. coli strains normally have the dehydrogenase. Those that do not willnot have the UDP-GlcA needed for HA synthesis, whereas those that havethe dehydrogenase (such as the K5 strain) have both sugar precursorsneeded for HA synthesis. When only the functional synthase was present,the K5 cells made HA, but the other strains did not (Table III). In nocase did recipient bacteria make HA without a functional HA synthasegene, HasA.

[0169] To make a bacterial strain overproduce HA one may place one ormore copies of both the HA synthase gene (and dehydrogenase gene ifdesired) into an appropriate recipient cell with functional promoters,ribosome binding sites etc. One would then find the best bacterialrecipient, gene copy number and mode of gene regulation.

[0170] At each stage of development one can construct one or severalbacterial strains containing the cloned HA synthase gene under thecontrol of different regulatory elements for expression of the gene.Constructs will also be made containing combinations of thedehydrogenase and synthase genes in various copy numbers. Thesebacterial strains will be tested in small scale fermentation trials. Onewould then increase the production scale by studying fermentationcultures first on a “bench-scale” (2 liters), then “pilot” (200 liters)and finally a “commercial” scale (15,000 liters).

[0171] The strains will be assessed for their growth characteristics andtheir ability to produce HA. The amount, size and stability of the HAwill be determined by standard testing procedures known to those ofskill in the art. There is significant interest in making the highest MWHA possible, since many biomedical applications for HA require thepolymer to be very long (high MW). It is likely that separate strainscan be constructed to achieve production of HA of different averagesizes.

[0172] Using Bacillus subtilis as a host cell offers distinct advantagesfor biotechnology and HA production. For example, the lack of endotoxinproduction by these cells is a big advantage in terms of FDA approvaland the ease of purifying the final product (vs Streptococcus). Thegenetics of B. subtilis has also been extensively studied. Furthermore,these cells make both sugar precursors needed for HA synthesis (Iwasaki,et al., 1989). The inventors have obsreved that the plasmid pPD41Δ5directs production of HA in B. subtilis strain 1A1 (with a productionrate of at least 500 mg/l of a culture having an OD₆₀₀ of 1).

[0173] This recombinant construct can be grown in a very simple andinexpensive growth media, such as Spizizen's media (2 gr. (NH₄)₂SO₄, 14gr. K₂HPO₄, 6 gr. KH₂PO₄, 1 gr. Sodium Citrate, and 5 gr. of glucose,per liter of water (Ausbel, et al., 1987)) supplemented with tryptophan(0.1 gr/ltr) and erythromycin (8 mg/ltr). On the other hand,Streptococcus bacteria must be grown on a more complex media that iseither expensive and/or contains large molecules that contaminate HApreparations from spent cultures. These results indicate that B.subtilis is a preferred host for the overproduction of HA. One canengineer a B. subtilis strain that produces a larger amount of HA thanis produced by streptococcal strains, because the latter may possess lowlevels of hyaluronidase, which degrade HA.

[0174] Therefore, initial efforts are to introduce the HA synthase geneinto an asporogenic strain of B. subtilis on a compatible plasmid, andalso by facilitated integration using methods described by others (Smithand Youngman, 1992; Prozorov, et al., 1987; Lewandoski and Smith, 1988;Ausubel, et al., 1987). One would then isolate cells containing variouscopies (say 1, 3 and 5) of the synthase gene and verify that they makeHA. The use of alternate promoters derived from B. subtilis can also bedetermined.

[0175] Based on the results of the production studies one can thenmodify or begin subsequent rounds of bacterial constructs. For example,one may decide to assess the effect of having different numbers of thesynthase gene and only one copy of the dehydrogenase gene. Thus, one can“fine-tune” the desired bacterial construct by successive testing andredesigning in order to optimize the quantity and quality of HAproduced.

[0176] All of the compositions and methods disclosed and claimed hereincan be made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe composition, methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

[0177] The following references, to the extent that they provideexemplary procedural or other details supplementary to those set forthherein, are specifically incorporated herein by reference.

[0178] Ausubel, F. M., et al. 1987. Current Prot. in Mol. Biol. Greene &Wiley-Intersci., NY.

[0179] Barson (1986), J. Pediatr. Orthop., 6:346-348.

[0180] Benjamin et al. (1976), J. Pediatr., 89:254:256.

[0181] Bitter, T. and Muir, H. (1962) Anal. Biochem. 4: 330-334.

[0182] Bulawa, C. E. (1992) Mol. Cell. Biol. 12: 1764-1776.

[0183] Caparon, M. G. and Scott, J. R. (1991) Meth. Enzymol. 204:556-586.

[0184] Clewell, D. B., Flannagan, S. E., Ike, Y., Jones, J. M., andGawron-Burke, C., (1988) J. Bacteriol. 170: 3046-3052.

[0185] Collins, C. H. and Lyne, P. M., (1976) Microbiological Methods,Butterworths, Boston, Mass., p.110.

[0186] Dao et al. (1985), Appl. Envir. Microbiol., 49:115-119.

[0187] DeAngelis, P. L., Papaconstantinou J., and Weigel, P. H. (1993a)J. Biol. Chem., 268:20, 14568-14571.

[0188] DeAngelis, P. L., Papaconstantinou J., and Weigel, P. H. (1993b)J. Biol. Chem., 268:26, 19181-19184.

[0189] Dinn (1971), J. Ir. Med. Assoc., 64:50-51.

[0190] Dochez, A. R., Avery, O. T., and Lancefield, R. C. (1919) J. Exp.Med. 30: 179-213.

[0191] Dougherty, B. A. and van de Rijn, I. (1992) J. Exp. Med. 175:1291-1299.

[0192] Dougherty, B. A. and van de Rijn, I. (1993) J. Biol. Chem.268:7118-7124.

[0193] Dunny, G. M., Lee, L. N. and LeBlanc, D. J. (1991) Appl. Environ.Microbiol. 57: 1194-1201.

[0194] DuPont Biotech. Update, 4, #4, July 1989.

[0195] Ehtesham, N. Z. and Hasnain, S. E. (1991) BioTechniques 11:718-721.

[0196] European Patent Application EP144019.

[0197] European Patent Application EP266578.

[0198] European Patent Application EP244757.

[0199] Evered, D. and Whelan, J. (eds.) 1989. The Biology of Hyaluronan.Wiley, Chichester, U.K.

[0200] Fiers et al., Nature, 273:113 (1978).

[0201] Gawron-Burke, C. and Clewell, D. B. (1984) J. Bacteriol. 159:214-221.

[0202] Gren, E. J. (1984) Biochimie 66:1-29.

[0203] Guerrero, R., Pedros-Alio, C., Schmidt, T. M., and Mas, J.(1985), Microbiologia, 1:53-65.

[0204] Gupta, D. S. Jann, B., Schmidt, G., Golecki, J. R., Orskov, I.,Orskov, F., and Jann, K., (1982) FEMS Microbiol. Letters 14:75-78.

[0205] H{dot over (a)}kansson S., and Holm S. E., (1986) Acta Path.Microbiol. Immunol. Scand. Sect. B 94:139-43.

[0206] Hirsch et al. (1960), J. Exp. Med., 111:309-322.

[0207] Ishimoto et al. (1967), Biochim. Biophys. Acta, 148:296-297.

[0208] Iwasaki, H., Araki, Y., Kaya, S. and Ito, E. (1989) Eur. J.Biochem. 178:635-641.

[0209] Jones, Genetics, 85:12 (1977).

[0210] Kass et al. (1944), J. Exp. Med., 79:319-330.

[0211] Kendall, F., Heidelberger, M., and Dawson, M. (1937) J. Biol.Chem. 118: 61-69.

[0212] Kyte & Doolittle (1982) J. Mol. Biol. 157:105-132

[0213] Laemmli, U. K. (1970) Nature 227: 680-685.

[0214] Lansing, M., Lellig, S., Mausolf, A., Martini, I., Crescenzi, F.,O'Regan, M., and Prehm, P. (1993) Biochem. J. 289: 179-184.

[0215] Laurent, T. C. and Fraser, J. R. E. (1992) FASEB J. 6: 2397-2404.

[0216] Lerouge, P., Roche, P., Faucher, C., Maillet, F., Truchet, G.,Prome, J. C., and Denarie, J. (1990) Nature 344: 781-784.

[0217] Lewandoski, M. and Smith, I. (1988) Plasmid 20:148-154.

[0218] MacLennan, A. P. 1956. J. Gen. Microbiol. 14:134-142.

[0219] Markovitz, A., Cifonelli, J. A., and Dorfman, A. (1959) J. Biol.Chem. 234: 2343-2350.

[0220] Markovitz et al. (1962), J. Biol. Chem., 237:273-279.

[0221] Matsumura, P., Silverman, M. and Simon, M. (1977) J. Bacteriol.132: 996-1002.

[0222] Maxam et al. (1980), Meth. Enzymol., 65:499.

[0223] Meagher, R. B., Tait, R. C., Betlach, M. and Boyer, H. W. (1977)Cell 10: 521-536.

[0224] Messing et al. (1981), Nucl. Acids Res., 9:309.

[0225] Min, H. and Cowman, M. K. (1986) Anal. Biochem. 155: 275-285.

[0226] Ng, K. F. and Schwartz, N. B. (1989) J. Biol. Chem. 264:11776-11783.

[0227] O'Connor, S. P. and Cleary, P. P. (1987) J. Infect. Dis. 156:495-504.

[0228] Prehm, P. (1983) Biochem. J. 211: 181-189.

[0229] Prehm, P. and Mausolf, A. 1986. Biochem. J. 235:887-889.

[0230] Prozorov, A. A., Poluektova, E. U., Savchenko, G. V.,Nezmetdinova, V. Z. and Khasanov, F. K. (1987) Gene 57: 221-227.

[0231] Quinn, A. W. and Singh, K. P., (1957) Biochem. J. 95:290-201.

[0232] Rizkallah et al. (1988), J. Infect. Dis., 158:1092-1094.

[0233] Rotta (1988), APMIS Suppl., 3:3-7.

[0234] Sambrook, J., Fritsch, E. F., and Maniatis, T., (1989) MolecularCloning: A Laboratory Manual. 2nd Ed., Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

[0235] Sanger et al. (1977), Proc. Natl. Acad. Sci. USA, 74:5463-5467.

[0236] Schaechter, M., Medoff, G., and Schlessinger, D., editors, (1989)Mechanisms of Microbial Disease, Williams and Wilkins, Baltimore, Md.

[0237] Scott et al. (1964), Histochemie, 4:73-85.

[0238] Siefkin et al. (1983), J. Clin. Microbiol., 17:386-388.

[0239] Smith, K. and Youngman, P. (1992) Biochimie 74: 705-711.

[0240] Stoolmiller et al. (1969), J. Biol. Chem., 244:236-246.

[0241] Studier et al. (1990), Meth. Enzymol. 185:60-89

[0242] Sugahara et al. (1979), J. Biol. Chem., 254:6252-6261.

[0243] Tengblad, A. (1980) Biochem. J. 185:101-105.

[0244] Tissue Culture, Academic Press, Kruse and Patterson, editors(1973).

[0245] Trieu-Cuot, P., Carlier, C., Poyart-Salmeron, C. and Courvalin,P. (1991) Gene 102:99-104.

[0246] Triscott, M. X. and van de Rijn, I. (1986) J. Biol. Chem. 261:6004-6009.

[0247] van de Rijn, I. and Drake, R. R. (1992) J. Biol. Chem. 267:24302-24306.

[0248] Wessels, M. R., Moses, A. E., Goldberg, J. B., and DiCesare, T.J., (1991) Proc. Natl. Acad. Sci. USA, 88:8317-8321.

[0249] Whitnack et al. (1981), Infect. Immun., 31:985-991.

1 9 1 1512 DNA Streptococcus pyogenes CDS (193)..(1449) 1 aattattttgggataattta tttaatatat attaaataaa ttatcctgat ttttcttttt 60 cgggggaatttttttaatgg aaacacaatt ttattaaaaa tatctctata tctagttgac 120 attatttcttatttatatta taatattgag gtcctttctt tcaaggaaat taaaaaagaa 180 agaggtgtaa ttgtg cct att ttt aaa aaa act tta att gtt tta tcc ttt 231 Val Pro Ile PheLys Lys Thr Leu Ile Val Leu Ser Phe 1 5 10 att ttt ttg ata tct atc ttgatt tat cta aat atg tat cta ttt gga 279 Ile Phe Leu Ile Ser Ile Leu IleTyr Leu Asn Met Tyr Leu Phe Gly 15 20 25 aca tca act gta gga att tat ggagta ata tta ata acc tat cta gtt 327 Thr Ser Thr Val Gly Ile Tyr Gly ValIle Leu Ile Thr Tyr Leu Val 30 35 40 45 atc aaa ctt gga tta tct ttc ctttat gag cca ttt aaa gga aat cca 375 Ile Lys Leu Gly Leu Ser Phe Leu TyrGlu Pro Phe Lys Gly Asn Pro 50 55 60 cat gac tat aaa gtt gct gct gta attcct tct tat aat gaa gat gcc 423 His Asp Tyr Lys Val Ala Ala Val Ile ProSer Tyr Asn Glu Asp Ala 65 70 75 gag tca tta tta gaa aca ctt aaa agt gtgtta gca cag acc tat ccg 471 Glu Ser Leu Leu Glu Thr Leu Lys Ser Val LeuAla Gln Thr Tyr Pro 80 85 90 tta tca gaa att tat att gtt gat gat ggg agttca aac aca gat gca 519 Leu Ser Glu Ile Tyr Ile Val Asp Asp Gly Ser SerAsn Thr Asp Ala 95 100 105 ata caa tta att gaa gag tat gta aat aga gaagtg gat att tgt cga 567 Ile Gln Leu Ile Glu Glu Tyr Val Asn Arg Glu ValAsp Ile Cys Arg 110 115 120 125 aac gtt atc gtt cac cgt tcc ctt gtc aataaa gga aaa cgc cat gct 615 Asn Val Ile Val His Arg Ser Leu Val Asn LysGly Lys Arg His Ala 130 135 140 caa gcg tgg gca ttt gaa aga tct gac gctgac gtt ttt tta acc gta 663 Gln Ala Trp Ala Phe Glu Arg Ser Asp Ala AspVal Phe Leu Thr Val 145 150 155 gac tca gat act tat atc tat cca aat gcctta gaa gaa ctc cta aaa 711 Asp Ser Asp Thr Tyr Ile Tyr Pro Asn Ala LeuGlu Glu Leu Leu Lys 160 165 170 agc ttc aat gat gag aca gtt tat gct gcaaca gga cat ttg aat gct 759 Ser Phe Asn Asp Glu Thr Val Tyr Ala Ala ThrGly His Leu Asn Ala 175 180 185 aga aac aga caa act aat cta tta acg cgactt aca gat atc cgt tac 807 Arg Asn Arg Gln Thr Asn Leu Leu Thr Arg LeuThr Asp Ile Arg Tyr 190 195 200 205 gat aat gcc ttt ggg gtg gag cgt gctgct caa tca tta aca ggt aat 855 Asp Asn Ala Phe Gly Val Glu Arg Ala AlaGln Ser Leu Thr Gly Asn 210 215 220 att tta gtt tgc tca gga cca ttg agtatt tat cga cgt gaa gtg att 903 Ile Leu Val Cys Ser Gly Pro Leu Ser IleTyr Arg Arg Glu Val Ile 225 230 235 att cct aac tta gag cgc tat aaa aatcaa aca ttc cta ggt tta cct 951 Ile Pro Asn Leu Glu Arg Tyr Lys Asn GlnThr Phe Leu Gly Leu Pro 240 245 250 gtt agc att ggg gat gat cga tgt ttaaca aat tat gct att gat tta 999 Val Ser Ile Gly Asp Asp Arg Cys Leu ThrAsn Tyr Ala Ile Asp Leu 255 260 265 gga cgc act gtc tac caa tca aca gctaga tgt gat act gat gta cct 1047 Gly Arg Thr Val Tyr Gln Ser Thr Ala ArgCys Asp Thr Asp Val Pro 270 275 280 285 ttc caa tta aaa agt tat tta aagcaa caa aat cga tgg aat aaa tct 1095 Phe Gln Leu Lys Ser Tyr Leu Lys GlnGln Asn Arg Trp Asn Lys Ser 290 295 300 ttt ttt aga gaa tct att att tctgtt aaa aaa att ctt tct aat ccc 1143 Phe Phe Arg Glu Ser Ile Ile Ser ValLys Lys Ile Leu Ser Asn Pro 305 310 315 atc gtt gcc tta tgg act att ttcgaa gtc gtt atg ttt atg atg ttg 1191 Ile Val Ala Leu Trp Thr Ile Phe GluVal Val Met Phe Met Met Leu 320 325 330 att gtc gca att ggg aat ctt ttgttt aat caa gct att caa tta gac 1239 Ile Val Ala Ile Gly Asn Leu Leu PheAsn Gln Ala Ile Gln Leu Asp 335 340 345 ctt att aaa ctt ttt gcc ttt ttatcc atc atc ttt atc gtt gct tta 1287 Leu Ile Lys Leu Phe Ala Phe Leu SerIle Ile Phe Ile Val Ala Leu 350 355 360 365 tgt cgt aat gtt cat tat atggtc aaa cat cct gct agt ttt ttg tta 1335 Cys Arg Asn Val His Tyr Met ValLys His Pro Ala Ser Phe Leu Leu 370 375 380 tct cct ctg tat gga ata ttacac ttg ttt gtc tta cag ccc cta aaa 1383 Ser Pro Leu Tyr Gly Ile Leu HisLeu Phe Val Leu Gln Pro Leu Lys 385 390 395 ctt tat tct tta tgc acc attaaa aat acg gaa tgg gga aca cgt aaa 1431 Leu Tyr Ser Leu Cys Thr Ile LysAsn Thr Glu Trp Gly Thr Arg Lys 400 405 410 aag gtc act att ttt aaataatatatgc atcgagtagt tagagaagga gtaatttt 1487 Lys Val Thr Ile Phe Lys415 atg aaa ata gca gtt gct gga tcag 1512 Met Lys Ile Ala Val Ala Gly420 425 2 419 PRT Streptococcus pyogenes 2 Val Pro Ile Phe Lys Lys ThrLeu Ile Val Leu Ser Phe Ile Phe Leu 1 5 10 15 Ile Ser Ile Leu Ile TyrLeu Asn Met Tyr Leu Phe Gly Thr Ser Thr 20 25 30 Val Gly Ile Tyr Gly ValIle Leu Ile Thr Tyr Leu Val Ile Lys Leu 35 40 45 Gly Leu Ser Phe Leu TyrGlu Pro Phe Lys Gly Asn Pro His Asp Tyr 50 55 60 Lys Val Ala Ala Val IlePro Ser Tyr Asn Glu Asp Ala Glu Ser Leu 65 70 75 80 Leu Glu Thr Leu LysSer Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu 85 90 95 Ile Tyr Ile Val AspAsp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu 100 105 110 Ile Glu Glu TyrVal Asn Arg Glu Val Asp Ile Cys Arg Asn Val Ile 115 120 125 Val His ArgSer Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp 130 135 140 Ala PheGlu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp 145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn 165 170175 Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg 180185 190 Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205 Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile LeuVal 210 215 220 Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile IlePro Asn 225 230 235 240 Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly LeuPro Val Ser Ile 245 250 255 Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala IleAsp Leu Gly Arg Thr 260 265 270 Val Tyr Gln Ser Thr Ala Arg Cys Asp ThrAsp Val Pro Phe Gln Leu 275 280 285 Lys Ser Tyr Leu Lys Gln Gln Asn ArgTrp Asn Lys Ser Phe Phe Arg 290 295 300 Glu Ser Ile Ile Ser Val Lys LysIle Leu Ser Asn Pro Ile Val Ala 305 310 315 320 Leu Trp Thr Ile Phe GluVal Val Met Phe Met Met Leu Ile Val Ala 325 330 335 Ile Gly Asn Leu LeuPhe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys 340 345 350 Leu Phe Ala PheLeu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn 355 360 365 Val His TyrMet Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu 370 375 380 Tyr GlyIle Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser 385 390 395 400Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr 405 410415 Ile Phe Lys 3 7 PRT Streptococcus pyogenes 3 Met Lys Ile Ala Val AlaGly 1 5 4 419 PRT Streptococcus pyogenes 4 Val Pro Ile Phe Lys Lys ThrLeu Ile Val Leu Ser Phe Ile Phe Leu 1 5 10 15 Ile Ser Ile Leu Ile TyrLeu Asn Met Tyr Leu Phe Gly Thr Ser Thr 20 25 30 Val Gly Ile Tyr Gly ValIle Leu Ile Thr Tyr Leu Val Ile Lys Leu 35 40 45 Gly Leu Ser Phe Leu TyrGlu Pro Phe Lys Gly Asn Pro His Asp Tyr 50 55 60 Lys Val Ala Ala Val IlePro Ser Tyr Asn Glu Asp Ala Glu Ser Leu 65 70 75 80 Leu Glu Thr Leu LysSer Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu 85 90 95 Ile Tyr Ile Val AspAsp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu 100 105 110 Ile Glu Glu TyrVal Asn Arg Glu Val Asp Ile Cys Arg Asn Val Ile 115 120 125 Val His ArgSer Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp 130 135 140 Ala PheGlu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp 145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn 165 170175 Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg 180185 190 Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205 Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile LeuVal 210 215 220 Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile IlePro Asn 225 230 235 240 Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly LeuPro Val Ser Ile 245 250 255 Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala IleAsp Leu Gly Arg Thr 260 265 270 Val Tyr Gln Ser Thr Ala Arg Cys Asp ThrAsp Val Pro Phe Gln Leu 275 280 285 Lys Ser Tyr Leu Lys Gln Gln Asn ArgTrp Asn Lys Ser Phe Phe Arg 290 295 300 Glu Ser Ile Ile Ser Val Lys LysIle Leu Ser Asn Pro Ile Val Ala 305 310 315 320 Leu Gln Thr Ile Phe GluVal Val Met Phe Met Met Leu Ile Val Ala 325 330 335 Ile Gly Asn Leu LeuPhe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys 340 345 350 Leu Phe Ala PheLeu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn 355 360 365 Val His TyrMet Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu 370 375 380 Tyr GlyIle Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser 385 390 395 400Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr 405 410415 Ile Phe Lys 5 7 PRT Streptococcus pyogenes 5 Met Lys Ile Ala Val AlaGly 1 5 6 16 DNA ARTIFICIAL SEQUENCE OLIGONUCLEOTIDE PROBE 6 aaagtgtgataagtcc 16 7 19 DNA ARTIFICIAL SEQUENCE OLIGONUCLEOTIDE PROBE 7tggcacaata tgtcagccc 19 8 19 DNA ARTIFICIAL SEQUENCE OLIGONUCLEOTIDEPROBE 8 tatggcttag tgccattcg 19 9 19 DNA ARTIFICIAL SEQUENCEOLIGONUCLEOTIDE PROBE 9 caatcatacc accaactgc 19

What is claimed is:
 1. A purified nucleic acid segment encoding HAsynthase, wherein the segment: (a) encodes the HA synthase of SEQ IDNO:2; or (b) is capable of hybridizing to the nucleotide sequence of SEQID NO:1 under standard hybridization conditions.
 2. The nucleic acidsegment of claim 1, further defined as including a nucleotide sequencein accordance with SEQ ID NO:1.
 3. The nucleic acid segment of claim 1,further defined as a recombinant vector.
 4. The nucleic acid segment ofclaim 3, wherein said recombinant vector is a plasmid.
 5. The nucleicacid segment of claim 4, further defined as an expression vectorcomprising a promoter operatively linked to the HA synthase codingregion.
 6. A nucleic acid segment which comprises at least a 14nucleotide long contiguous stretch which corresponds to, or iscomplementary to, a nucleic acid sequence of SEQ ID NO:1, and is capableof hybridizing to said nucleic acid segment under standard hybridizationconditions.
 7. The nucleic acid segment of claim 6, further defined ascomprising at least a 20 nucleotide long contiguous stretch whichcorresponds to, or is complementary to, the nucleic acid sequence of SEQID NO:1.
 8. The nucleic acid segment of claim 7, further defined ascomprising at least a 30 nucleotide long contiguous stretch whichcorresponds to, or is complementary to, the nucleic acid sequence of SEQID NO:1.
 9. The nucleic acid segment of claim 8, further defined ascomprising at least a 50 nucleotide long contiguous stretch whichcorresponds to, or is complementary to, the nucleic acid sequence of SEQID NO:1.
 10. The nucleic acid segment of claim 9, further defined ascomprising at least a 100 nucleotide long contiguous stretch whichcorresponds to, or is complementary to, the nucleic acid sequence of SEQID NO:1.
 11. The nucleic acid segment of claim 10, further defined ascomprising at least a 200 nucleotide long contiguous stretch whichcorresponds to, or is complementary to, the nucleic acid sequence of SEQID NO:1.
 12. The nucleic acid segment of claim 11, further defined ascomprising at least a 300 nucleotide long contiguous stretch whichcorresponds to, or is complementary to, the nucleic acid sequence of SEQID NO:1.
 13. The nucleic acid segment of claim 12, further defined ascomprising at least a 500 nucleotide long contiguous stretch whichcorresponds to, or is complementary to, the nucleic acid sequence of SEQID NO:1.
 14. The nucleic acid segment of claim 13, further defined ascomprising at least a 1000 nucleotide long contiguous stretch whichcorresponds to, or is complementary to, the nucleic acid sequence of SEQID NO:1.
 15. The nucleic acid segment of claim 14, further defined ascomprising at least a 1441 nucleotide long contiguous stretch whichcorresponds to, or is complementary to, the nucleic acid sequence of SEQID NO:1.
 16. The nucleic acid segment of claim 15, further defined ashaving the nucleic acid sequence of SEQ ID NO:1.
 17. The nucleic acidsegment of claim 6, further defined as comprising a nucleic acidfragment of up to 10,000 basepairs in length.
 18. The nucleic acidsegment of claim 17, further defined as comprising a nucleic acidfragment of up to 5,000 basepairs in length.
 19. The nucleic acidsegment of claim 18, further defined as comprising a nucleic acidfragment of up to 3,000 basepairs in length.
 20. The nucleic acidsegment of claim 19, further defined as comprising a nucleic acidfragment of up to 1,000 basepairs in length.
 21. The nucleic acidsegment of claim 20, further defined as comprising a nucleic acidfragment of up to 500 basepairs in length.
 22. The nucleic acid segmentof claim 21, further defined as comprising a nucleic acid fragment of upto 100 basepairs in length.
 23. The nucleic acid segment of claim 6,further defined as a DNA segment.
 24. The nucleic acid segment of claim6, further defined as including a detectable label.
 25. A recombinanthost cell comprising the recombinant vector having a DNA segment inaccordance with claim 1 or claim
 10. 26. The recombinant host cell, ofclaim 25, wherein the host cell is a prokaryotic cell.
 27. Therecombinant host cell of claim 26, wherein the host cell is a eukaryoticcell.
 28. A purified composition comprising a polypeptide having anamino acid sequence in accordance with SEQ ID NO:2.
 29. A method ofdetecting a DNA species comprising the steps of: (a) obtaining a DNAsample; (b) contacting said DNA sample with a nucleic acid segment inaccordance with SEQ ID NO:1 under conditions effective to allowhybridization to form a complex; and (c) detecting said complex.
 30. Amethod for detecting a bacterial cell that expresses hyaluronic acidsynthase, comprising the steps of: (a) obtaining a bacterial cell samplesuspected of expressing hyaluronan synthase; (b) contacting nucleicacids from said cell sample with a nucleic acid segment in accordancewith SEQ ID NO:1 under conditions effective to allow hybridization ofsubstantially complementary nucleic acid sequences; and (c) identifyingthe presence of hybridized complexes containing substantiallycomplementary nucleic acid sequences within said sample; wherein thepresence of increased levels of said substantially complementary nucleicacid sequence is indicative of a bacterial strain that expresseshyaluronan synthase.
 31. The method of claim 29, wherein the nucleicacids contacted are located within said cell.
 32. The method of claim29, wherein the nucleic acids are separated from said cell prior tocontact.
 33. The method of claim 29, wherein the nucleic acid segmentcomprises a detectable label and hybridized complexes are detected bydetecting said label.
 34. The method of claim 30, wherein said nucleicacid segment comprises a radio-, an enzymatic-, a fluorescent-, abiotinyl-, or a chemiluminescent-label.
 35. A method of preparinghyaluronic acid comprising: (a) providing a recombinant host cellbearing a vector that encodes a protein that includes an amino acidsequence in accordance with SEQ ID NO:2; (b) culturing said cells underconditions permitting nucleic acid expression and protein production;and (c) recovering hyaluronic acid so produced.
 36. The method of claim35, wherein said host cell is a prokaryote.
 37. The method of claim 36,wherein said host cell is a Streptococcal, Lactococcal, Bacilli,Salmonella, Enterococcus, or E. coli host.
 38. The method of claim 35,wherein said host cell is a eukaryote.
 39. The method of claim 38,wherein said host cell is a Saccharomyces cell.
 40. The method of claim38, wherein said host cell is a HeLa, Vero, NIH 3T3, Chinese HamsterOvary, or a COS cell.
 41. The method of claim 38, wherein said host cellis an insect cell.
 42. The method of claim 41, wherein said host cell isinfected with a recombinant baculovirus.